Suppressed neurogenesis without cognitive deficits: effects of fast neutron irradiation in mice.

This study assessed the effects of combined low-dose neutron and γ-ray irradiation on hippocampal neurogenesis and hippocampal-dependent memory. Neural progenitor cell division and survival were evaluated in brain sections and whole hippocampal preparations following head irradiation at a dose of 0.34 Gy for neutron radiation and 0.36 Gy for γ-ray radiation. Hippocampal-dependent memory formation was tested in a contextual fear conditioning task following irradiation at doses of 0.4 Gy for neutron radiation and 0.42 Gy for γ-ray radiation. Cell division was suppressed consistently along the entire dorsoventral axis of the hippocampus 24 h after the irradiation, but quiescent stem cells remained unaffected. The control and irradiated mice showed no differences in terms of exploratory behavior or anxiety 6 weeks after the irradiation. The ability to form hippocampus-dependent memory was also unaffected. The data may be indicative of a negligible effect of the low-dose of fast neutron irradiation and the neurogenesis suppression on animal behavior at 6 weeks after irradiation.

Cerebral impact of prenatal irradiation by 131I: an experimental model of clinical neuroradioembryological effects. / VPLYV PRENATAL'NOGO OPROMINENNIa 131I NA GOLOVNYY̆ MOZOK: EKSPERYMENTAL'NA MODEL' KLINIChNYKh NEY̆RORADIOEMBRIOLOGIChNYKh EFEKTIV.

Human brain in prenatal period is a most vulnerable to ionizing radiation body structure. Unlike atomic bombings or radiological interventions in healthcare leading at most to external irradiation the intensive internal exposure may occur upon nuclear reactor accidents followed by substantial release and fallout of radioactive 131I. The latter can lead to specific neuroradioembryological effects. OBJECTIVE: To create an experimental model of prenatal cerebral radiation effects of 131I in human and to determine the experimental and clinical neuroradioembryological effects.Study object. The neuroradioembryological effects in Vistar rats exposed to 131I in prenatal period. Nervous system status and mental status in 104 persons exposed to ionizing radiation in utero due to the ChNPP accident and the same in 78 not exposed subjects. METHODS: Experimental i.e. behavioral techniques, including the spontaneous locomotive, exploratory activity and learning ability assessment, clinical i.e. neuropsychiatric, neuro and psychometric, neuropsychological, neurophys iological methods, both with dosimetric and statistical methods were applied. RESULTS: Intrauterine irradiation of Wistar rats by 131I was simulated on a model of one time oral 27.5 kBq radionu clide administration in the mid gestation period (0.72±0.14 Gy fetal thyroid dose), which provides extrapolation of neuroradioembryological effects in rats to that in humans exposed to intrauterine radiation as a result of the Chornobyl catastrophe. Abnormalities in behavioral reactions and decreased output of conditioned reflex reactions identified in the 10 month old rats suggest a deterioration of cerebral cognition in exposed animals. Specific cog nitive deficit featuring a disharmonic intellectual development through the relatively decreased verbal intelligence versus relative increase of nonverbal one is remained in prenatally exposed persons. This can indicate to dysfunc tion of cortical limbic system with especial involvement of a dominant hemisphere hippocampus. Decreased theta band spectral power (4-7 Hz range) of cerebral bioelectrical activity in the left frontotemporal area is suggestive of hippocampal dysfunction mainly in dominant hemisphere of prenatally irradiated persons. Disorders of hippocam pal neurogenesis due to prenatal exposure by radioactive iodine can be a biologic basis here. Innovative approach es in social adaptation, psychoprophylaxis and psychorehabilitation involve the maximum effective application and development of just the most developed psychological and cognitive abilities in survivors.

Alpha1-adrenergic receptor blockade in the VTA modulates fear memories and stress responses.

Activity of the ventral tegmental area (VTA) and its terminals has been implicated in the Pavlovian associative learning of both stressful and rewarding stimuli. However, the role of the VTA noradrenergic signaling in fear responses remains unclear. We aimed to examine how alpha1-adrenergic receptor (α1-AR) signaling in the VTA affects conditioned fear. The role of α1-AR was assessed using the micro-infusions into the VTA of the selective antagonists (0.1-1µg/0.5µl prazosin and 1µg/0.5µl terazosin) in acquisition and expression of fear memory. In addition, we performed control experiments with α1-AR blockade in the mammillary bodies (MB) - a brain region with α1-AR expression adjacent to the VTA. Intra-VTA but not intra-MB α1-AR blockade prevented formation and retrieval of fear memories. Importantly, local administration of α1-AR antagonists did not influence footshock sensitivity, locomotion or anxiety-like behaviors. Similarly, α1-AR blockade in the VTA had no effects on negative affect measured as number of 22kHz ultrasonic vocalizations during fear conditioning training. We propose that noradrenergic signaling in the VTA via α1-AR regulates formation and retrieval of fear memories but not other behavioral responses to stressful environmental stimuli. It enhances the encoding of environmental stimuli by the VTA to form and retrieve conditioned fear memories and to predict future behavioral outcomes. Our results provide novel insight into the role of the VTA α1-AR signaling in the regulation of stress responsiveness and fear memory.

Pontomesencephalic Tegmental Afferents to VTA Non-dopamine Neurons Are Necessary for Appetitive Pavlovian Learning.

The ventral tegmental area (VTA) receives phenotypically distinct innervations from the pedunculopontine tegmental nucleus (PPTg). While PPTg-to-VTA inputs are thought to play a critical role in stimulus-reward learning, direct evidence linking PPTg-to-VTA phenotypically distinct inputs in the learning process remains lacking. Here, we used optogenetic approaches to investigate the functional contribution of PPTg excitatory and inhibitory inputs to the VTA in appetitive Pavlovian conditioning. We show that photoinhibition of PPTg-to-VTA cholinergic or glutamatergic inputs during cue presentation dampens the development of anticipatory approach responding to the food receptacle during the cue. Furthermore, we employed in vivo optetrode recordings to show that photoinhibition of PPTg cholinergic or glutamatergic inputs significantly decreases VTA non-dopamine (non-DA) neural activity. Consistently, photoinhibition of VTA non-DA neurons disrupts the development of cue-elicited anticipatory approach responding. Taken together, our study reveals a crucial regulatory mechanism by PPTg excitatory inputs onto VTA non-DA neurons during appetitive Pavlovian conditioning.

Memory formation and retrieval of neuronal silencing in the auditory cortex.

Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior.

[Influence of transcranial electromagnetic brain stimulation on development of conditioned reflex in rats].

The influence of transcranial electromagnetic stimulation on the development of an active avoidance reflex with painful reinforcement in laboratory rats is investigated. It is shown, that an exposure of the rats' brain to electromagnetic radiation in the millimeter range ((λ = 5,6 and 7,1 mm), modulated as a series of low-frequency pulses, leads to a suppression of the development of the conditioned avoidance reflex occurred in 50% of cases. In other 25% of cases irradiation leads to inhibition of reflex development. Transcranial electromagnetic stimulation after intraperitoneal injection of the blocking agent of serotonergic receptors (kitryl) has no influence on reflex development. Electromagnetic brain stimulation does not influence reflex retention in the case when it has been acquired. Based on the data obtained it is assumed that transcranial electromagnetic stimulation promotes the development of serotonin, exerting an inhibiting effect on the formation of temporal bindings of the studied conditioned reflex.

Cranial irradiation regulates CREB-BDNF signaling and variant BDNF transcript levels in the mouse hippocampus.

The brain can be exposed to ionizing radiation in various ways, and such irradiation can trigger adverse effects, particularly on learning and memory. However, the precise mechanisms of cognitive impairments induced by cranial irradiation remain unknown. In the hippocampus, brain-derived neurotrophic factor (BDNF) plays roles in neurogenesis, neuronal survival, neuronal differentiation, and synaptic plasticity. The significance of BDNF transcript variants in these contexts is becoming clearer. In the present study, both object recognition memory and contextual fear conditioning task performance in adult C57BL/6 mice were assessed 1 month after a single exposure to cranial irradiation (10 Gy) to evaluate hippocampus-related behavioral dysfunction following such irradiation. Furthermore, changes in the levels of BDNF, the cAMP-response element binding protein (CREB) phosphorylation, and BDNF transcript variants were measured in the hippocampus 1 month after cranial irradiation. On object recognition memory and contextual fear conditioning tasks, mice evaluated 1 month after irradiation exhibited significant memory deficits compared to sham-irradiated controls, but no apparent change was evident in locomotor activity. Both phosphorylated CREB and BDNF protein levels were significantly downregulated after irradiation of the hippocampus. Moreover, the levels of mRNAs encoding common BDNF transcripts, and exons IIC, III, IV, VII, VIII, and IXA, were significantly downregulated after irradiation. The reductions in CREB phosphorylation and BDNF expression induced by differential regulation of BDNF hippocampal exon transcripts may be associated with the memory deficits evident in mice after cranial irradiation.

ApoE isoform modulates effects of cranial 56Fe irradiation on spatial learning and memory in the water maze.

Apolipoprotein E, which plays an important role in lipid transport and metabolism and neuronal repair, might modulate the CNS risk following (56)Fe irradiation exposure during space missions. In this study, we investigated this risk by behavioral and cognitive testing male E2, E3, and E4 mice 3 months following cranial (56)Fe irradiation. In the open field, mice irradiated with 2 Gy showed higher activity levels than sham-irradiated mice or mice irradiated with 1 Gy. In addition, E2 mice showed higher activity and lower measures of anxiety than E3 and E4 mice in the open field and elevated zero maze. During hidden platform training, sham-irradiated mice showed most robust learning, 1 Gy irradiated mice reduced learning, and 2 Gy irradiated mice no improvement over the four sessions. In the water maze probe trials, sham-irradiated E2, E3, and E4 mice and E2 and E4 mice irradiated with 1 Gy showed spatial memory retention, but E3 mice irradiated with 1 Gy, and E2, E3, and E4 mice irradiated with 2 Gy did not. Thus, cranial (56)Fe irradiation increases activity levels in the open field and impairs spatial learning and memory in the water maze. E3 mice are more susceptible than E2 or E4 mice to impairments in spatial memory retention in the water maze, indicating that apoE isoform modulates the CNS risk following space missions.

Light enhances learned fear.

The ability to learn, remember, and respond to emotional events is a powerful survival strategy. However, dysregulated behavioral and physiological responses to these memories are maladaptive. To fully understand learned fear and the pathologies that arise during response malfunction we must reveal the environmental variables that influence learned fear responses. Light, a ubiquitous environmental feature, modulates cognition and anxiety. We hypothesized that light modulates responses to learned fear. Using tone-cued fear conditioning, we found that light enhances behavioral responses to learned fear in C57BL/6J mice. Mice in light freeze more in response to a conditioned cue than mice in darkness. The absence of significant freezing during a 2-wk habituation period and during intertrial intervals indicated that light specifically modulates freezing to the learned acoustic cue rather than the context of the experimental chamber. Repeating our assay in two photoreceptor mutant models, Pde6b(rd1/rd1) and Opn4(-/-) mice, revealed that light-dependent enhancement of conditioned fear is driven primarily by the rods and/or cones. By repeating our protocol with an altered lighting regimen, we found that lighting conditions acutely modulate responses when altered between conditioning and testing. This is manifested either as an enhancement of freezing when light is added during testing or as a depression of freezing when light is removed during testing. Acute enhancement, but not depression, requires both rod/cone- and melanopsin-dependent photoreception. Our results demonstrate a modulation by light of behavioral responses to learned fear.

Effects of the SARM ACP-105 on rotorod performance and cued fear conditioning in sham-irradiated and irradiated female mice.

Female mice are more susceptible to radiation-induced cognitive changes than male mice. Previously, we showed that, in female mice, androgens antagonize age-related cognitive decline in aged wild-type mice and androgens and selective androgen receptor modulators (SARMs) antagonize cognitive changes induced by human apolipoprotein E4, a risk factor for developing age-related cognitive decline. In this study, the potential effects of the SARM ACP-105 were assessed in female mice that were either sham-irradiated or irradiated with ¹³7Cesium at a dose of 10Gy. Behavioral testing started 2 weeks following irradiation. Irradiation impaired sensorimotor function in vehicle-treated mice but not in ACP-105-treated mice. Irradiation impaired cued fear conditioning and ACP-105 enhanced fear conditioning in sham-irradiated and irradiated mice. When immunoreactivity for microtubule-associated protein 2 was assessed in the cortex of sham-irradiated mice, there was a brain area × ACP-105 interaction. While ACP-105 reduced MAP-2 immunoreactivity in the sensorimotor cortex, there was a trend towards increased MAP-2 immunoreactivity in the enthorhinal cortex. No effect on MAP-2 immunoreactivity was seen in the irradiated cortex or sham-irradiated or irradiated hippocampus. Thus, there are relatively early radiation-induced behavioral changes in female mice and reduced MAP-2 levels in the sensorimotor cortex following ACP-105 treatment might contribute to enhanced rotorod performance.

Arrest of adult hippocampal neurogenesis in mice impairs single- but not multiple-trial contextual fear conditioning.

The role of adult hippocampal neurogenesis in contextual fear conditioning (CFC) is debated. Several studies demonstrated that blocking adult hippocampal neurogenesis in rodents impairs CFC, while several other studies failed to observe an impairment. We sought to determine whether different CFC methods vary in their sensitivity to the arrest of adult neurogenesis. Adult neurogenesis was arrested in mice using low-dose, targeted x-irradiation, and the effects of irradiation were assayed in conditioning procedures that varied in the use of a discrete conditioned stimulus, the number of trials administered, and the final level of conditioning produced. We demonstrate that irradiation impairs CFC in mice when a single-trial CFC procedure is used but not when multiple-trial procedures are used, regardless of the final level of contextual fear produced. In addition, we show that the irradiation-induced deficit in single-trial CFC can be rescued by providing preexposure to the conditioning context. These results indicate that adult hippocampal neurogenesis is required for CFC in mice only when brief training is provided.

Involvement of the lateral septal area in the expression of fear conditioning to context.

Considering the evidence that the lateral septal area (LSA) modulates defensive responses, the aim of the present study is to verify if this structure is also involved in contextual fear conditioning responses. Neurotransmission in the LSA was reversibly inhibited by bilateral microinjections of cobalt chloride (CoCl(2), 1 mM) 10 min before or after conditioning or 10 min before re-exposure to the aversively conditioned chamber. Only those animals that received CoCl(2) before re-exposure showed a decrease in both cardiovascular and behavioral conditioned responses. These results suggest that the LSA participates in the expression, but not acquisition or consolidation, of contextual fear conditioning.

Sex-dependent effects of 56Fe irradiation on contextual fear conditioning in C57BL/6J mice.

Effects of irradiation on hippocampal function have been mostly studied in male rodents and relatively little is known about potential effects of irradiation on hippocampal function in female rodents. Moreover, although the long-term effects of clinical radiation on cognitive function have been well established, the effects of other forms of irradiation, such as high charged, high energy radiation (HZE particles) that astronauts encounter during space missions have not been well characterized. In this study we compared the effects of (56)Fe irradiation on fear conditioning in C57BL/6J female and male mice. Hippocampus-dependent contextual fear conditioning was impaired in female mice but improved in male mice following (56)Fe irradiation. Such impairment was not seen for hippocampus-independent cued fear conditioning. Thus, the effects of (56)Fe irradiation on hippocampus-dependent contextual fear conditioning are critically modulated by sex.

Ionizing radiation impairs the formation of trace fear memories and reduces hippocampal neurogenesis.

Long-term cognitive impairments are a feared consequence of therapeutic cranial irradiation in children as well as adults. Studies in animal models suggest that these deficits may be associated with a decrease in hippocampal granule cell proliferation and survival. In the present study the authors examined whether whole brain irradiation would affect trace fear conditioning, a hippocampal-dependent task. Preadolescent (postnatal Day 21, PD 21), adolescent (PD 50), and postadolescent (PD 70) rats received single doses of 0 Gray (Gy), 0.3 Gy, 3 Gy, or 10 Gy whole brain irradiation. Three months after radiation treatment, a significant dose-dependent decrease in bromo-deoxyuridine positive cells was observed. Irradiation produced a dose-dependent decrease in freezing in response to the conditioned stimulus in all age groups. Interestingly, the authors found no differences in context freezing between irradiated and control groups. Further, there were no differences in delay fear memories, which are independent of hippocampus function. Our results strongly indicate that irradiation impairs associative memories dependent on hippocampus and this deficit is accompanied by a decrease in granule cell neurogenesis indicating that these cells may be involved in normal hippocampal memory function.

Mechanisms underlying functional changes in the primary motor cortex ipsilateral to an active hand.

Performance of a unimanual hand motor task results in functional changes in both primary motor cortices (M1(ipsilateral) and M1(contralateral)). The neuronal mechanisms controlling the corticospinal output originated in M1(ipsilateral) and the resting hand during a unimanual task remain unclear. Here, we assessed functional changes within M1(ipsilateral) and in interhemispheric inhibition (IHI) associated with parametric increases in unimanual force. We measured motor-evoked potential (MEP) recruitment curves (RCs) and short-interval intracortical inhibition (SICI) in M1(ipsilateral), IHI from M1(contralateral) to M1(ipsilateral), and the influence of IHI over SICI using transcranial magnetic stimulation at rest and during 10, 30, and 70% of maximal right wrist flexion force. EMG from the left resting flexor carpi radialis (FCR) muscle was comparable across conditions. Left FCR MEP RCs increased, and SICI decreased with increasing right wrist force. Activity-dependent (rest and 10, 30, and 70%) left FCR maximal MEP size correlated with absolute changes in SICI. IHI decreased with increasing force at matched conditioned MEP amplitudes. IHI and SICI were inversely correlated at increasing forces. In the presence of IHI, SICI decreased at rest and 70% force. In summary, we found activity-dependent changes in (1) SICI in M1(ipsilateral), (2) IHI from M1(contralateral) to M1(ipsilateral), and (3) the influence of IHI over SICI in the left resting hand during force generation by the right hand. Our findings indicate that interactions between GABAergic intracortical circuits mediating SICI and interhemispheric glutamatergic projections between M1s contribute to control activity-dependent changes in corticospinal output to a resting hand during force generation by the opposite hand.

Modulatory effect of ionizing radiation on food-NaCl associative learning: the role of gamma subunit of G protein in Caenorhabditis elegans.

Ionizing radiation (IR) is known to impair learning by suppressing adult neurogenesis in the hippocampus. However, in a mature nervous system, IR-induced functional alterations that are independent of neurogenesis remain largely unknown. In the present study, we analyzed the effects of IR on a food-NaCl associative learning paradigm of adult Caenorhabditis elegans that does not undergo neurogenesis. We observed that a decrease in chemotaxis toward NaCl occurs only after combined starvation and exposure to NaCl. Exposure to IR induced an additional decrease in chemotaxis immediately after an acute dose in the transition stage of the food-NaCl associative learning. Strikingly, chronic irradiation induced negative chemotaxis in the exposed animals, i.e., the primary avoidance response. IR-induced additional decreases in chemotaxis after acute and chronic irradiation were significantly suppressed in the gpc-1 mutant, which was defective in GPC-1 (one of the two gamma subunits of the heterotrimeric G-protein). Chemotaxis to cAMP, but not to lysine and benzaldehyde, was influenced by IR during the food-NaCl associative learning. Our novel findings suggest that IR behaves as a modulator in the food-NaCl associative learning via C. elegans GPC-1 and a specific neuronal network and may shed light on the modulatory effect of IR on learning.

[Dynamics of the rats higher nervous activity disturbances after partial influence of high energy electrons in high doses].

The dynamics of the disturbances of stabilized motor defensive conditioned reflex of active avoidance in "shuttle-box" in rats after total and partial (the head or body irradiated) influence of high energy electrons in dose 100 Gy was investigated. The head irradiation, the same way as total irradiation, provoked the early effects of disturbances of higher nervous activity, specifically, initial shock-like effect--the "early transient incapacitation" (ETI). The head shielding, on the contrary, prevents these disturbances. At the same times the body irradiation in dose 10 Gy (this dose don't provoke ETI effect) provoked practically the same disturbances of higher nervous activity as the total irradiation. Consequently, in animals irradiated in super-lethal doses the early disturbances of higher nervous activity provoked of direct influence of ionizing radiation to the brain. In animals irradiated in lesser doses these disturbances caused of non direct effects basically.

The lateral amygdala processes the value of conditioned and unconditioned aversive stimuli.

The amygdala is critical for acquiring and expressing conditioned fear responses elicited by sensory stimuli that predict future punishment, but there is conflicting evidence about whether the amygdala is necessary for perceiving the aversive qualities of painful or noxious stimuli that inflict primary punishment. To investigate this question, rats were fear conditioned by pairing a sequence of auditory pips (the conditioned stimulus, or CS) with a brief train of shocks to one eyelid (the unconditioned stimulus, or US). Conditioned responding to the CS was assessed by measuring freezing responses during a test session conducted 24 h after training, and unconditioned responding to the US was assessed by measuring head movements evoked by the eyelid shocks during training. We found that pre-training electrolytic lesions of the amygdala's lateral (LA) nucleus blocked acquisition of conditioned freezing to the CS, and also significantly attenuated unconditioned head movements evoked by the US. Similarly, bilateral inactivation of the amygdala with the GABA-A agonist muscimol impaired acquisition of CS-evoked freezing, and also attenuated US-evoked responses during training. However, when amygdala synaptic plasticity was blocked by infusion of the NR2B receptor antagonist ifenprodil, acquisition of conditioned freezing was impaired but shock reactivity was unaffected. These findings indicate that neural activity within the amygdala is important for both predicting and perceiving the aversive qualities of noxious stimuli, and that synaptic plasticity within LA is the mechanism by which the CS becomes associated with the US during fear conditioning.

[The effect of pulsed cyclical microware radiation on the conditioned behavior of rats]. / Vliianie tsiklicheskogo impul'snogo mikrovolnovogo oblucheniia na uslovno-reflektornuiu deiatel'nost' krys.

Research has been carried out to investigate the effects of pulsed cyclical microware exposure (7 GHz, 400 pps, 100 mcs, 10-20 mW/cm2, 10 or 20 cycles of "5 min exposure--4 min pause") on avoidance learning of rats. It was shown that reductions in conditioned behavior after cyclical pulsed microware exposure occurred at an SAR of 2.1 W/kg (10 mW/cm2). It was found the cumulation of the effects of the cycles at prolonged cyclical microwave exposures.

Neural correlates of Pavlovian conditioning in components of the neural network supporting ciliary locomotion in Hermissenda.

Pavlovian conditioning in Hermissenda consists of pairing light, the conditioned stimulus (CS) with activation of statocyst hair cells, the unconditioned stimulus (US). Conditioning produces CS-elicited foot shortening and inhibition of light-elicited locomotion, the two conditioned responses (CRs). Conditioning correlates have been identified in the primary sensory neurons (photoreceptors) of the CS pathway, interneurons that receive monosynaptic input from identified photoreceptors, and putative pedal motor neurons. While cellular mechanisms of acquisition produced by the synaptic interaction between the CS and US pathways are well-documented, little is known about the mechanisms responsible for the generation or expression of the CR. Here we show that in conditioned animals light reduced tonic firing of ciliary activating pedal neurons (VP1) below their pre-CS baseline levels. In contrast, pseudorandom controls expressed a significant increase in CS-elicited tonic firing of VP1 as compared to pre-CS baseline activity. Identified interneurons in the visual pathway that have established polysynaptic connections with VP1 were examined in conditioned animals and pseudorandom controls. Depolarization of identified type Ie interneurons with extrinsic current elicited a significant increase in IPSPs recorded in VP1 pedal neurons of conditioned animals as compared with pseudorandom controls. Conditioning also enhanced intrinsic excitability of type Ie interneurons of conditioned animals as compared to pseudorandom controls. Light evoked a modest increase in IPSP frequency in VP1 of conditioned preparations and a significant decrease in IPSP frequency in VP1 of pseudorandom controls. Our results show that a combination of synaptic facilitation and intrinsic enhanced excitability in identified components of the CS pathway may explain light-elicited inhibition of locomotion in conditioned animals.