Up-regulation of gamma-aminobutyric acid transporter I mediates ethanol sensitivity in mice.

Ethanol is among the most widely abused drugs in the world. Chronic ethanol consumption leads to ethanol tolerance and addiction, and impairs learning and memory. Na+/Cl- dependent GABA transporters play an important role in controlling the concentration of GABA in the synaptic cleft, and thus they control the intensity and duration of synaptic transmission of GABA. It has been suggested that GABAergic system is involved in ethanol consumption, tolerance and addiction, because chronic ethanol consumption alters the expression of GABAA receptors and drugs on GABA receptors affect ethanol actions. The results of the present study reveal that that activity of GABA transporters in mouse brain after 15-min acute ethanol injection or after chronic ethanol consumption is increased. Moreover, mice pre-injected with a competitive or a noncompetitive antagonist of gamma-aminobutyric acid transporter subtype 1 (GAT1) showed high sensitivity to the sedative/hypnotic effects of ethanol. In contrast, transgenic mice overexpressing GAT1 displayed low sensitivity to ethanol, as shown by the righting reflex test. Mice overexpressing GAT1 survived a lethal dose of ethanol (9 g/kg, i.p.) longer, maintained locomotor activity longer after a sub-lethal dose (1.75 g/kg, i.p.) and exhibited a higher median lethal dose than wild-type littermates. These results suggest that GAT1 plays an important role in sensitivity to ethanol, and might be a therapeutic target for alcoholism prevention and treatment. Acute and chronic ethanol administration resulted in the increase of GABA transporter function. Use of GAT1 selective inhibitors and GAT1 overexpressing mice thus demonstrate that GAT1 should be an important protein mediating sensitivity to ethanol in mice.

Overexpression of gamma-aminobutyric acid transporter subtype I leads to cognitive deterioration in transgenic mice.

AIM: To explore the physiological role of gamma-aminobutyric acid transporter subtype I (GAT1) in cognition. METHODS: Transgenic mice were produced by pronuclei microinjection method. Integration of transgene was identified by Southern-blot and PCR analysis in various generations. Level of GAT1 mRNA in a variety of tissues was evaluated by semi-quantitative RT-PCR analysis. GAT1 protein was detected by immunofluorescence and histochemistry analysis. Associative learning capacity was analyzed by conditioned avoidance task. Memory retention was assessed by novel object recognition test. Morphology of synaptosomes was examined by electron microscope. RESULTS: Four independent founder mice bearing various copies of transgene were generated. GAT1 was evidently overexpressed at both mRNA and protein level in a variety of tissues from transgenic mice. In comparison with wild-type mice, transgenic mice exhibited significantly declined associative learning capacity (P < 0.01) and decreased memory retention (P < 0.01 in 1-h-retention, and P < 0.05 in 1-d-retention). In addition, the amount of asymmetric synapses in the brain of transgenic mice was reduced approximately by 24 %, relative to wild-type mice. CONCLUSION: Overexpression of GAT1 in mice results in cognitive deterioration, indicating that the alteration in the expression of gamma-aminobutyric acid transporters is involved in the pathophysiological mechanism underlying some cognitive deficiencies.

Overexpression of gamma-aminobutyric acid transporter subtype I leads to susceptibility to kainic acid-induced seizure in transgenic mice.

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

Gamma-aminobutyric acid transporter (GAT1) overexpression in mouse affects the testicular morphology.

Gamma-aminobutyric acid and GABAergic receptors were previously reported to be distributed in reproductive systems besides CNS and predicted to participate in the modulation of testicular function. Gamma-aminobutyric acid transporter was implicated to be involved in this process. However, the potential role of gamma-aminobutyric transporter in testis has not been explored. In this study, we investigated the existence of mouse gamma-aminobutyric acid transporter subtype I (mGAT1) in testis. Wild-type and transgenic mice, which overexpressing mGAT1 in a variety of tissues, especially in testis, were primarily studied to approach the profile of mGAT1 in testis. Mice with overexpressed mGAT1 develop normally but with reduced mass and size of testis as compared with wild-type. Testicular morphology of transgenic mice exhibited overt abnormalities including focal damage of the spermatogenic epithelium accompanied by capillaries proliferation and increased diameter of seminiferous tubules lumen. Reduced number of spermatids was also found in some seminiferous tubules. Our results clearly demonstrate the presence of GAT1 in mouse testis and imply that GAT1 is possibly involved in testicular function.

Transgenic mice overexpressing gamma-aminobutyric acid transporter subtype I develop obesity.

Transgenic mice ubiquitously overexpressing murine gamma-aminobutyric acid transporter subtype I were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgenic mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. This preliminary finding indicates that the inappropriate level of gamma-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.

Alpha-2 adrenergic modulation of prefrontal cortical neuronal activity related to spatial working memory in monkeys.

The effects of systemically administered or iontophoretically applied clonidine (alpha-2 adrenergic agonist) and iontophoretically applied yohimbine (alpha-2 adrenergic antagonist) were examined on prefrontal cortical (PFC) neurons related to spatial working memory (SWM). Systemically administered clonidine (0.04 mg/kg) enhanced SWM-related PFC neuronal activity by 32.5 +/- 14.5%, (mean +/- SD; n = 25 neurons). The facilitatory effect of clonidine was antagonized by iontophoretically applied yohimbine. Iontophoretically applied clonidine enhanced SWM-related PFC neuronal activity by 38.2 +/- 18.6%, (n = 13 neurons), whereas similarly applied yohimbine suppressed it by 34.4 +/- 17.8% (n = 28 neurons). These results indicate that: a) systemically administered clonidine can facilitate SWM-related PFC neuronal activity through actions at alpha-2 adrenoceptors in the PFC; and b) conversely, blockade by yohimbine of alpha-2 adrenoceptors in the PFC suppresses SWM-related neuronal activity. The present study provides neurophysiological evidence that alpha-2 adrenoceptors in the PFC are involved in the cellular mechanisms underlying working memory.

[A microdialysis study of excitatory amino acid levels of the monkeys' caudate nucleus during the delayed go/no-go task].

Using in vivo microdialysis coupled with HPLC-fluorescent detection chemical analysis method, levels of excitatory amino acid were monitored in the caudate nucleus of rhesus monkeys (Macaca mulatta) during performance of a delayed go/no-go task in which movement or no-movement responses were executed depending on the position of a cue light. It was found that the levels of glutamate and aspartate in caudate microdialysates decreased by (31.68 +/- 3.85)% (n = 10, t9 = 6.51, P < 0.001) and (26.25 +/- 5.95)% (n = 10, t9 = 3.39, P < 0.01) respectively during the delayed go/no-go task performance as compared to their basal levels before the task performance. Glutamine and asparagine levels were also found decreased significantly (P < 0.05). In contrast, no such decreases were seen when the monkey performing a non-delayed go/no-go task or performing the task composed of delayed go-trials only. The results suggest that the excitatory amino acid transmission in the caudate nucleus may be involved in the delayed go/no-go task performance, and thereby provide a direct evidence for the modulation of motor working memory by glutamatergic transmission in caudate nucleus.

[Intracerebral microdialysis and its application in neurosciences].

As a new technique for sampling the extracellular fluid in vivo, the microdialysis is recently attractive to neuroscientists. Coupled with high sensitivity techniques for chemical microanalysis, it has been successfully used for monitoring chemical changes in extracellular environment of intact brain, and applied to some fields of neuroscience studies. In this article, its principle and some methodological aspects were described systematically, and a brief overview of its applications in neurosciences and some new advances was also given. Besides, the scope and limitations of microdialysis was discussed in the light of our own experience.

Delayed-response deficit induced by local injection of the alpha 2-adrenergic antagonist yohimbine into the dorsolateral prefrontal cortex in young adult monkeys.

Two young adult monkeys (Macaca mullata) were trained to perform a delayed-response task that required the monkeys to remember a cued spatial position (left or right) over a delay interval and then to make a response to the cued position. Local injection of the alpha 2-adrenergic antagonist yohimbine (10 micrograms in 2 microliters saline) into the dorsolateral prefrontal cortex (Walker's area 46 and area 9) impaired the performance of the delayed-response task, and it was without effect on the performance of the task if there was no delay between the cue and choice signals. The main performing error after injection of yohimbine was that the monkeys responded to uncued position with higher rate. Local injection of the alpha 1-adrenergic antagonist prazosin (10 microgram in 2 microliters saline) or the beta-adrenergic antagonist propranolol (10 micrograms in 2 microliters saline) into the same cortical areas induced no significant effect on the performance of the task. The present study suggests that prefrontal alpha 2-adrenoceptors play an important role in the spatial working memory in young adult monkeys.

[Prefrontal injection of the alpha 2-adrenergic antagonist yohimbine impairs performance of a delayed GO/NO-GO task in monkeys].

Two rhesus monkeys were trained to perform a delayed GO/NO-GO task, which was initiated by the monkey's pressing a lever. At 0.5 s after the lever-pressing, a white square (1.5 cm x 1.5 cm) was displayed on the right or the left of a computer screen and lasted 0.5 s as a cue. After a delay of 0.0-4.0 s, a red square (1.5 cm x 1.5 cm) appeared between the left-cue and right-cue positions as a signal for response choice. The monkey released the lever within 0.8 s if the cue had been on the right (GO trial), or kept on the lever-pressing for 1.2 s if the cue had been on the left (NO-GO trial). Short-term working memory during the delay was a non-spatially representational one. Local injection of the alpha 2-adrenergic antagonist yohimbine (10 micrograms) into the prefrontal cortex (Walker's areas 46, 9) contralateral to the performing hand decreased the correct rate of performance. The longer the delay period was, the more serious the impairment was. The performing error after injection of yohimbine was expressed mainly as "commissural" response (the monkey made GO response in NO-GO trial or NO-GO response in GO trial). Injection of the alpha 1-adrenergic antagonist prazosin (10 micrograms) or the beta-adrenergic antagonist propranolol (10 micrograms) did not impair the task-performance. Our results suggest that, prefrontal alpha 2-adrenoceptors may play an important role in the short-term working memory with non-spatial representation.

The role of monosialoganglioside GM1 in LTP-induction in rat hippocampal slices.

The effect of monosialoganglioside GM1 of different doses on the long-term potentiation (LTP) of synaptic transmission has been studied in the CA1 region of rat hippocampal slices, and the possible role that calcium ion and NMDA receptor play has also been investigated. The results reveal that larger magnitude of LTP is induced in hippocampal slices pre-incubated with GM1. The dose-response curve appears in diphase, and the largest magnitude of LTP has been obtained at the GM1 concentration of 50 mg/L in incubation ACSF. Moreover, the magnitude of LTP induced from the slices pre-incubated with GM1 at lower calcium ion concentration is similar to that obtained from the control slices at normal calcium ion concentration. Under higher calcium ion concentration, the enhancing effect of GM1 on LTP seems relatively feeble. After NMDA receptors were blocked, no enhancing effect of GM1 was observed. The mechanism of GM1 action on LTP is discussed.

The mandibular swing-transcervical approach to the skull base: anatomical study. Technical note.

This report describes in a stepwise fashion the surgical anatomy of an approach to the midline and lateral compartments of the skull base (clivus, infralabyrinthine/infratemporal regions). The salient features of this procedure are represented by a mandibulotomy and by detachment of the pharynx from the skull base through a combined oral and cervical approach. There is full neurovascular control of the internal carotid artery and lower cranial nerves with the possibility of complete exposure of the intrapetrous and intracavernous segments of the internal carotid artery on the side of the exposure. This approach, which may be regarded as an expansion of the original work of Krespi, should be considered when dealing aggressively with extensive skull-base lesions invading the midline and lateral compartments of the skull base.

Effects of exogenous gangliosides on learning and memory in rats.

After 10 or more consecutive daily injections (I.P. 50 mg/kg for developing, adult and aged, 2.5 mg/mouse for neonatal) of total bovine gangliosides, rats were trained to perform the discrimination tasks. The results show that gangliosides can enhance the learning ability and memory retention in rats at different ages.

[The role of GABA-ergic transmission of caudate-putamen nucleus (CPN) in the control of conditioned behavior in rats].

Using the method of bilateral injection of GABA (100 micrograms/microliters) and muscimol (0.1 micrograms/microliters) into the caudate-putamen nucleus (CPN) we have studied the role of GABA-ergic transmission of CPN in the control of conditioned behavior. In order to compare the effects of GABA and muscimol on two behavioral paradigms, rats of either sex weighing 160-180 g were trained to perform discrimination learning and conditioned avoidance response. A tone (1 kHz, 80 dB) was used as a conditioned stimulus. After bilateral injections of GABA and muscimol into the trained rats' CPN respectively, the conditioned avoidance response was inhibited temporarily, whereas the discrimination learning remained almost unchanged. In addition, the inhibitory effect of muscimol on the conditioned avoidance response was attenuated or even abolished by following bilateral intra-CPN injection of picrotoxin (0.1 micrograms/microliters for each side). As a control, the same volume of saline was injected into CPN bilaterally, and it showed that there was no change of both the conditioned avoidance response and the discrimination learning. The results show that the GABA-ergic transmission of CPN does play a role in the control of avoidance conditioning in rats, and thus support the suggestion that GABA-ergic transmission of the caudate-globus pallidus plays an important role in the control of conditioned behavior.

Multibarreled glass-coated tungsten microelectrode for both neuronal activity recording and iontophoresis in monkeys.

A multibarreled glass-coated tungsten microelectrode suitable for single-unit recording and iontophoresis in chronically behaving animals is described. The microelectrode was stiff enough to pass through the dura matter of behaving monkeys, and can be applied as a microiontophoretic electrode. The electrode is easy to build and usable in stabilized conditions after repeated penetrations.

[Comparison between responses of monkey prefrontal neurons to signal and to indifferent stimuli].

Unit activities of the principal sulcus area were recorded in three monkeys (Macaca mullata). Two of them were trained to perform visual discrimination task, and the other had not received any training. The sequence of the task is as follows: (1) a cue of presentation of red (in R Model) or blue (in B Model) light (1.3s); (2) a delay period (2.1s); (3) a reaction period of presentation of white light (4.88s). The monkey should draw a bar after the onset of the white light of R Model to avoid a electric shock, and should not draw the bar after the onset of the white light of B Model in which the monkeys would not receive the electric shock. Of 127 prefrontal units recorded from the two trained monkeys when they were performing the task, 80 units (63%) were related to the events of the task. Ninety six units were recorded for the test of responses to click and flash in the intertrial interval, and 23 units (24%) responded to click and/or flash. Fifteen out of 23 units were event-related. Twelve out of 103 units responded to the red, blue and white flash in the intertrial interval. A hundred units were recorded from the untrained monkey a only 4 units showed nonspecific responses to the indifferent stimuli. The results showed that after training much more prefrontal neurons involved in processing of the information of signal stimuli than those in response to the indifferent stimuli. In other words, the prefrontal neurons possess plasticity and change their activities during the training of visual discrimination task.

[Investigation of cortical slow potentials during the delayed discrimination learning in monkeys].

Averaged slow potentials (SPs) were recorded at different training stages of delayed discrimination learning in two monkeys. The details of the task sequence was described in our previous work. It was found that at the early stages of the training the amplitude of SPs was very small, and it increased gradually with the increase of percentage of correct responses. The SPs recorded from the prefrontal cortex had the largest amplitude and there was no SPs recorded from the rear portion of the cortex. SPs recorded from the symmetrical sites of the two hemispheres showed no significant differences in amplitude and duration. Moreover, the SPs did not show significant differences in amplitude and distribution between two test models (R Model and B Model). The results obtained from the extinction experiments showed that after extinction of the behavioral responses the SPs disappeared. The results showed that the SPs recorded were learning-dependent and represented the association of events. As the SPs appeared in the delay period and were prefrontal-dominant, it was suggested that the prefrontal cortex might play an important role in short-term memory process.