Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities.

Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV's putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV(-/-)) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV(+/-)) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV(-/-) and PV(+/-) mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a common link between apparently unrelated ASD-associated synapse structure/function phenotypes.

Self-administration of small amounts of morphine through glass micropipettes into the ventral tegmental area of the rat.

A micropipette-system was used to investigate intracranial self-injection of morphine in the rat. The system consisted of a glass micropipette (tip dia 5 microns) connected by flexible tubing and a swivel to a pressure source. Using nose-poking through a hole in one wall of the cage as the operant, rats self-injected quantities as small as 50 ng/5 nl of morphine into the ventral tegmental area. In contrast, rats in the saline-treated group and yoked-control group did not increase their rates of nose-poking behaviour above baseline levels. Intermittent reinforcement in the morphine-treated group resulted in a further increase in the rate of nose-poking. Histological inspection of the site of injection revealed minimal damage to brain tissue and likely confinement of the injected solution to the area around the tip of the pipette.

The effects of long-term nicotine treatment on locomotion, exploration and memory in young and old rats.

To assess the effects of long-term treatment with nicotine on several behavioral measures (locomotor activity, exploratory efficiency, habituation, short-term and long-term memory) of young (5 months) and old (22 months) rats in a hexagonal tunnel maze, nicotine was added to the drinking water (0, 20 or 50 mg/l) for up to 131 experimental days. With the exception of effects on exploratory efficiency, young and old rats did not differ in their response to the drug. Nicotine decreased body weight throughout the experiment. Nicotine treatment reduced water intake during the first 30 min of the daily 4.5 h access to drinking water. Nicotine increased locomotor activity throughout the experiment. When nicotine treatment was discontinued during a 7-day withdrawal period, locomotor activity immediately dropped to control values. Intertrial habituation was not affected by nicotine. Long-term nicotine treatment had an attenuating effect on exploratory efficiency in young rats; however, the drug did not influence performance in tasks measuring spatial memory. Finally, age increased weight, decreased locomotor activity and impaired exploratory efficiency and short-term memory. Age, however, did not affect the performance of the long-term memory task.

Facilitation of tunnel maze performance by systemic injection of the neurokinin substance P.

The effect of peripheral injections of substance P (SP) on performance in two different configurations of an automated tunnel maze was examined in three experiments. In two experiments, the effect of pretrial SP injections (10-1000 micrograms/kg) on performance in the hexagonal and radial maze configuration of an automated tunnel maze was investigated. In the hexagonal maze, which measures activity, exploratory efficiency, habituation, and perimeter walking, injection of SP affected perimeter walking only. In the radial maze, SP produced a facilitation of measures of efficiency and long-term and short-term memory without affecting activity. In the third experiment, the effect of pre- and posttrial injections of SP (50 or 500 micrograms/kg) on performance in the radial maze configuration was tested. Again, pretrial injections of 500 micrograms of SP facilitated performance with respect to measures of efficiency and short- and long-term memory; 50 micrograms produced a weaker effect. Virtually no effect was seen with posttrial injections.

Modification of the David-Kopf puller (DKI 700 C) for the preparation of multi-barrel glass micropipettes.

The DKI vertical pipette puller (Model 700 C), which is normally used to make single or multi-barrel glass pipettes out of prepulled blanks, can be modified for the construction of a variety of multi-barrel pipettes. The use of the modified pipette puller is described for the production of small diameter 4-barrel pipettes.

Intracranial application of substances in the unrestrained, awake rat by pressure injection through glass micropipettes.

A method is described which allows intracranial injection of drug solutions through glass micropipettes (tip diameter 15 micron) in the freely moving rat by use of air pressure. Compared with conventional injection methods through steel cannulae this method has the advantages of (1) minimal destruction of brain tissue, (2) precise injection of small volumes of solutions (1 nl to several microliters), (3) simultaneous recording of local EEG activity, and (4) usage of multipipette assemblies.

Effects of CGS 19755 and dizocilpine (MK 801) on delayed time discrimination performance.

The effects of the competitive NMDA (N-methyl-D-aspartate) receptor antagonist CGS 19755 and the non-competitive NMDA receptor antagonist dizocilpine (MK 801) on time discrimination and short-term memory were investigated in rats trained on a delayed time discrimination task. In a two-lever operant chamber, pressing one lever was correct and reinforced with a food pellet after presentation of a stimulus light for 2 s (SD(short)); pressing the opposite lever was correct after presentation of a stimulus light for 8 s (SD(long)). CGS 19755 (3.0 mg/kg) attenuated performance, decreased nose-pokes (an activity necessary to trigger the presentation of the discriminative stimulus and the presentation of the response levers), and increased response latencies (time from 'opportunity to leverpress' to 'actual leverpress'). The highest dose of dizocilpine (0.2 mg/kg) tested also attenuated performance. Further, the number of nose-pokes and response latencies were not altered by any dose of dizocilpine. With increasing delays, saline-injected animals developed a bias towards reporting an occurrence of an SD(long), independent of the actual stimulus presented. This bias was attenuated or even reversed by CGS 19755 (3.0 mg/kg) and (0.2 mg/kg). Our results suggest that NMDA receptors are directly or indirectly involved in time discrimination performance.

Water maze performance and hippocampal weight of prenatally stressed rats.

Prenatally stressed rats were tested for water maze performance with the water temperature kept at 18 degrees C (low stress) or cooled down to 12 degrees C (high stress). When the platform had been removed from the pool and the water was kept at 12 degrees C, prenatally stressed males--but not females--spent more time searching for the platform in the correct quadrant of the pool than their controls. Prenatal stress reduced hippocampal weight in both sexes.

Conditioned taste aversion as a learning and memory paradigm.

Conditioned taste aversion (CTA) is a well established learning and memory paradigm in rats and mice that is considered to be a special form of classical conditioning. Rodents--as well as many other species including man--learn to associate a novel taste (CS) with nausea (US), and as a consequence avoid drinking fluid with this specific taste. In contrast to other types of classical conditioning, even CS-US intervals lasting several hours lead to an aversion to the gustatory CS. With increasing CS-US delay duration, however, the aversion against the CS gradually decreases. Mice differ from rats in their reaction to the CS as well as the US. They tolerate a much higher concentration of saccharin and they do not show any clear signs of nausea when injected with the US. Advantages of this task are its relative independence of motor behavior, well described pathways for the CS and partly the US, and the wealth of available anatomical and pharmacological data implying several brain structures (e.g. parabrachial nucleus, amygdala, insular cortex), neurotransmitters and their receptors (e.g. cholinergic system, NMDA-receptors), and cellular processes (e.g. expression of immediate early genes, Ras-MAP kinase signaling pathway, CREB phosphorilation, protein tyrosine phosphorilation, protein synthesis) in CTA. The CTA paradigm has also been successfully used to phenotype mouse mutants.

The effect of continuous intraventricular infusion of L1 and NCAM antibodies on spatial learning in rats.

Recent studies suggest a role of the neural cell adhesion molecules L1 and NCAM in mechanisms of memory storage. In the present study we analyzed the effect of continuous intraventricular infusion of polyclonal antibodies directed against L1 (antiL1) or NCAM (antiNCAM) on the performance of male Wistar rats during the acquisition and retention of a spatial learning task (Morris water-maze). In this task animals have to learn the spatial position of a hidden escape platform in a water tank to escape onto it. During acquisition of the task animals with continuous infusion of antiNCAM - but not those infused with antiL1 - showed day-dependent attenuated learning in comparison to controls (P = 0.001). Control animals were either injected with vehicle (PBS) or with polyclonal antibodies raised against liver cell membrane. When the escape platform was removed during the retention test (transfer test), the performance of animals continuously infused with antiL1 as well as those continuously infused with antiNCAM showed an impaired search pattern when compared with the performance of control animals (P = 0.001 and 0.04, respectively). Whereas control animals spent up to 46% of their time searching for the platform in the correct quadrant, the time antiL1- and antiNCAM-infused animals spent in this quadrant was closer to chance level (30.5% and 36.5%), respectively). The present data provide additional support for an involvement of the two adhesion molecules L1 and NCAM in synaptic plasticity underlying memory storage.

Circadian activity, nociceptive thresholds, nigrostriatal and mesolimbic dopaminergic activity in the Naples High- and Low-Excitability rat lines.

These experiments were designed to further characterize the differential phenotypic constellation of the Naples High- (NHE) and Naples Low-Excitability (NLE) lines. In order to determine possible differences between NHE and NLE rats in activity and circadian rhythms, besides reactivity to novelty (selection trait), adult male rats of both strains were tested during two 10-min exposures to a Làt-maze. They were then kept in activity cages continuously for 3 days. Moreover, nociceptive thresholds were measured with the hot-plate and the tail-flick test, to probe the possibility that these rats could be differentially sensitive to nociceptive stimuli. Further, the integrity of the nigro-striatal and mesolimbic system was investigated by measuring tyrosine-hydroxylase activity in the striatum and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum as well as in the nucleus accumbens. In addition, TH activity was measured in the adrenals to probe the sympathetic section of the neurovegetative system. The results indicate that NHE and NLE rats differ by a factor of two in their phasic activity in a Làt-maze. In contrast, no differences in 24-h activity during the dark or light phase could be observed in the activity cages. However, NHE rats anticipated the light-on stimulus in the morning by reducing their activity 1 h earlier than NLE rats. Further, no difference could be found with the hot-plate and the tail-flick test. Finally, biochemical analyses revealed no difference in the NHE and NLE rats in the main terminal zone of mesolimbic system (n. accumbens) nor of nigrostriatal system (striatum) nor in the adrenal glands. In conclusion, since the only consistent difference between NHE and NLE rats appears to be reactivity to spatial novelty, an hippocampus-dependent behavioral trait (selection trait), independent of altered activity in the sympathetic system or dopaminergic activity in the major dopaminergic brain systems, the usefulness of these strains as genetic model to test current hypotheses of spatial processor device(s) in the mammalian brain is supported.

Unilateral intranigral injection of MPTP in the rat induces contraversive turning.

Unilateral injection of MPTP into the pars compacta of the substantia nigra in rats induced contraversive turning immediately after the injection. Contraversive turning decreased, and reversed its direction after about 30 min. Ipsiversive turning was still present 24 h after the injection of MPTP. These results suggest that MPTP has an initial stimulatory effect on dopaminergic neurons followed by a depression of the activity of these cells.

Non-lateralization of a temporal discrimination task acquired under unilateral cortical spreading depression.

Forty-nine rats were trained for three days to obtain water from a drinking tube for every 15th lick. On the next day 44 of them learned under unilateral cortical spreading depression to lick for differential reinforcement of low rates of responding (minimum interlick interval 6 sec - DRL 6). On day 5 the DRL 6 task was retrained with either the contralateral (n = 32) or ipsilateral (n = 12) cortical hemisphere depressed. Five rats served as unoperated controls. In all three groups DRL performance was significantly improved on day 5. The failure to achieve DRL lateralization by functional hemidecortication suggests that DRL responding has a strong non-neocortical component.

Sensitization of the perioral biting reflex by intranigral GABA agonist after detelencephalization.

Unilateral injection of 160 ng (0.4 microliter) of muscimol into the substantia nigra pars reticulata of rats elicits contraversive turning and simultaneously sensitizes the perioral biting reflex on the side of the face contralateral to the injected substantia nigra. Bilateral removal of the telencephalon eliminated neither the muscimol-induced behavior nor the peroral biting reflex.

Distributed changes in c-Fos and c-Jun immunoreactivity in the rat brain associated with arousal and habituation to novelty.

The effects of exposure to spatial novelty on expression of the immediate early gene (IEG) products c-Fos and c-Jun were mapped in the rat brain by immunohistochemistry. Adult male Sprague-Dawley rats were tested for 10 min in a Làt-maze, and corner-crossings, rearings, and fecal boli were recorded. Rats were sacrificed at different time intervals after exposure to the maze (0.5, 2, 6, or 24 h). Unexposed rats or rats repeatedly exposed for 3 days at 24 h interval served as controls. Nonperfused brains were processed for immunocytochemistry for c-Fos and c-Jun on adjacent slices using the avidin-biotin method and diaminobenzidine as chromogen. In unexposed control rats the constitutive expression of the two IEGs products was low and scattered. In contrast, rats that had been exposed for the first time to the maze (spatial novelty) showed an extensive c-Fos- and c-Jun-like immunoreactivity in the reticular formation, the caudate-putamen complex, the hippocampus (granular and pyramidal neurons), the cerebellum (granular neurons), and all layers of somatosensory cortex. The positivity was stronger in rats exposed for the first time to the box than in repeatedly exposed or unexposed control rats. A maximal IEG expression was found in animals with postexposure survival times of 2 and 6 h. IEG expression in repeatedly exposed rats was still above baseline expression of unexposed rats but still lower than that of rats having been exposed only once to the maze.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation and lateralization of sensorimotor field for perioral biting reflex by intranigral GABA agonist and by systemic apomorphine in the rat.

In Experiment 1 unilateral injection of the GABA agonist, muscimol (20 ng in 0.1 microliter saline) into the substantia nigra (SN) of rats elicited contraversive turning and a correlated transient asymmetry in responsiveness to tactile stimulation of the mouth area. On the side of the face contralateral to the injected SN they responded stronger than on the ipsilateral side, and, furthermore, they responded to tactile stimulation with a withdrawal of the lip, followed by a vigorous biting of the probe. In Experiment 2, systemic injection of apomorphine also sensitized the perioral biting response to tactile stimulation. Unilateral injection into the SN of 8 micrograms 6-OHDA blocked this response to stimulation of the side of the face ipsilateral to the lesion. The experiments demonstrate: (1) pharmacological control of sensory-motor neglect, the asymmetry being determined by the direction of turning; and (2) a possible neuropharmacological basis of the perioral biting reflex, which may be related to certain types of aggressive behaviors.

Immediate early genes and brain DNA remodeling in the Naples high- and low-excitability rat lines following exposure to a spatial novelty.

The aim of these studies was to map the neural consequences of exposure to a spatial novelty on the expression of immediate gene (IEG) and on unscheduled brain DNA synthesis (UBDS) in two genetic models of altered activity and hippocampal functions, i.e., the Naples High- (NHE) and Low-excitability (NLE) rats. Adult male rats of NLE and NHE lines, and of a random-bred stock (NRB) were tested in a Làt-maze, and corner crossings, rearings, and fecal boli were counted during two 10-min tests 24 h apart. For IEG expression, rats were exposed to a Làt-maze with nonexposed or repeatedly exposed rats used as controls, and were sacrificed at different time intervals thereafter. For UBDS, rats were sacrificed immediately after the first or the second exposure o a Làt-maze. IEG expression was measured by immunocytochemistry for the FOS and JUN proteins. NRB rats exposed for the first time to the maze showed extensive FOS and JUN positive cells in the reticular formation, the granular and pyramidal neurons of hippocampus, the amygdaloid nuclei, all layers of somatosensory cortex, and the granule cells of the cerebellar cortex. The positivity, stronger in rats exposed for the first time, was present between 2 and 6 h and was prevented by the NMDA receptor antagonist CPP (5 mg/kg). The positivity was very low in NHE rats, and it was stronger in NLE compared to NRB rats. UBDS was measured in ex vivo homogenates of brain areas by the incorporation into DNA of 3H-[methyl]-thymidine given intraventricularly 15 min before test trial 1 or 2 (pulse of 0.5 h).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of anticholinergic drugs on delayed time discrimination performance in rats.

To investigate the effects of the muscarinic antagonist scopolamine and the nicotinic antagonist mecamylamine on time discrimination and short-term memory, rats were trained on a delayed conditional time discrimination task until performance stabilized. In a two-lever operant chamber, pressing one lever was correct after the presentation of a stimulus light for 2 seconds (SD short); and pressing the other lever was correct after presentation of a stimulus light for 8 seconds (SD long). Scopolamine (0.06 mg, 0.25 mg, and 1.0 mg/kg) attenuated performance in a dose-dependent manner. Furthermore, the drug decreased nose-pokes (an activity necessary to trigger the presentation of the discriminative stimuli and the presentation of the response levers), and increased response delay (time from opportunity to lever press to actual lever press). Performance attenuating effects of mecamylamine in the time discrimination task did not appear unless high doses (8 mg/kg) of the drug were given. With increasing delays between 0 and 8 seconds, animals injected with saline developed a bias towards reporting the occurrence of the SD long, independent of the actual stimulus presented. A reversal of this bias was seen in animals injected with scopolamine; they more often reported the occurrence of the SD short. Our results support a role for muscarinic receptors in discrimination learning, attention, and time estimation.

The effects of acute and chronic stress on motor and sensory performance in male Lewis rats.

Any behavioral testing induces stress to some degree. A meaningful interpretation of behavioral results can be difficult if stress, caused by handling or the testing situation, modifies the experimental outcome. Especially for neurological animal models, it is important to know how stress affects motor and sensory performance. Therefore, we investigated the effects of varying degrees of stress on several motor and sensory tasks that are frequently used to assess functional recovery after lesion-induced impairments in adult rats. Acute, subchronic, and chronic stress impaired ladder walking and prolonged the duration of grasping a bar. Stress also altered walking patterns by increasing the base of support and foot rotation and reducing stride length. Furthermore, chronic stress induced hypersensitivity to painful stimuli, but did not significantly influence the latency to remove sticky papers from the hindpaws (sticky paper test). In the light--dark (L/D) test, stress reduced the latency to enter the dark compartment and enhanced the number of transitions supporting that cold swim stress modifies the animal's level of anxiety. These data point towards a critical influence of acute or chronic stress on motor control and sensory performance of rats, suggesting that stress might be a critical intervening variable of the outcome of behavioral tests.

Prenatal stress in rats: effects on plasma corticosterone, hippocampal glucocorticoid receptors, and maze performance.

The present experiments were designed to investigate the effects of maternal stress on cognitive and endocrine parameters in the adult offspring. Pregnant rats were stressed daily during the last week of pregnancy (days 15-19) by restraint, and the performance of their offspring in the Morris water maze was recorded. Plasma corticosterone levels after swimming and the status of hippocampal glucocorticoid receptors (GRs) were determined. During acquisition of the task, prenatally stressed (PS) males - but not females - showed longer escape latencies than non-stressed controls when swimming in cold (10 degrees C) but not in warm (20 degrees C) water. This sex- and prenatal stress-specific difference was even more pronounced during reversal learning of the task. In contrast, PS females - but not males - had higher basal corticosterone levels and a lower density of hippocampal corticosteroid receptors than non-stressed controls. In all animals irrespective of treatment, swimming in the water maze causes an increase of corticosterone that was smaller on day 8 of swimming than on day 1. After swimming in cold water, the rise in corticosterone levels in females was steeper and returned faster to baseline values than after swimming in warm water. A similar pattern could be seen in PS females when compared to their non-stressed controls. The data suggest that prenatal stress impairs spatial learning in males but not in females. Basal and stress-induced increases in corticosterone levels, however, were altered in PS females and not in PS males; i.e., prenatal stress-induced changes in corticosterone secretion were not paralleled by prenatal stress-induced deficits in spatial learning.