Deficient cerebellar long-term depression, impaired eyeblink conditioning, and normal motor coordination in GFAP mutant mice.

Mice devoid of glial fibrillary acidic protein (GFAP), an intermediate filament protein specifically expressed in astrocytes, develop normally and do not show any detectable abnormalities in the anatomy of the brain. In the cerebellum, excitatory synaptic transmission from parallel fibers (PFs) or climbing fibers (CFs) to Purkinje cells is unaltered, and these synapses display normal short-term synaptic plasticity to paired stimuli in GFAP mutant mice. In contrast, long-term depression (LTD) at PF-Purkinje cell synapses is clearly deficient. Furthermore, GFAP mutant mice exhibited a significant impairment of eyeblink conditioning without any detectable deficits in motor coordination tasks. These results suggest that GFAP is required for communications between Bergmann glia and Purkinje cells during LTD induction and maintenance. The data support the notion that cerebellar LTD is a cellular mechanism closely associated with eyeblink conditioning, but is not essential for motor coordination tasks tested.

Acute neural damage in the rat neocortex in vitro induced by a combination of anoxia and mechanical stress.

To elucidate the mechanisms of neural damage after brain ischemia, rat neocortical slices were exposed to anoxia at room temperature for 1 h, and other slices were prepared from the neocortical blocks exposed to anoxia at room temperature for 1 h. Field potentials elicited by the stimulation of layer IV were recorded in supragranular layers in these slices. No clear damage was observed electrophysiologically or morphologically in these slices. In contrast, a complete loss of the trans-synaptic field potentials and a decrease in the density of the cells stained with Neutral Red were elicited by injecting an anoxic medium into the neocortical blocks at room temperature for 1 h. In the slice preparations, the injection of the anoxic medium failed to reproduce clear neural damage, while a combination of mechanical stress and anoxia elicited a complete loss of trans-synaptic potentials; this was alleviated by Gd3+ (50 microM) and D(-)-2-amino-5-phosphonovaleric acid (100 microM). These results indicate that a combination of mechanical stress and anoxia produces acute and severe neural damage even at room temperature in vitro. The mechanism of the damage and the relationship between the neural damage in vitro and in vivo are discussed.

Cerebellar long-term potentiation under suppressed postsynaptic Ca2+ activity.

To study the roles of postsynaptic Ca2+ activity in cerebellar synaptic plasticity, we used a patch-recording technique in Purkinje cell dendrites. While the combination of parallel fibre stimulation and 8-bromo cyclic guanosine monophosphate (Br-cGMP) application produced long-term depression (LTD) of the parallel fibre/Purkinje cell transmission, the same stimuli evoked long-term potentiation (LTP) during postsynaptic injection of Ethyleneglycol-bis-(beta-amino-ethylether)-N, N, N', N'-tetraacetate (EGTA). Furthermore, in the presence of alpha-aminobutyric acid (GABA), parallel fibre stimulation plus Br-cGMP produced LTP of extracellular K+ increases following parallel fibre stimulation. These results suggest that postsynaptic Ca2+ activity in Purkinje cells is negatively correlated to the direction of plastic changes and that the Ca2+ changes and cGMP play distinct roles in cerebellar synaptic plasticity.

Cerebrospinal fluid nitric oxide metabolites in painful diseases.

To elucidate the involvement of NO in pain transmission in humans, we measured NO metabolites (nitrite/nitrate) in the CSF of patients with painful diseases using an NO analyzer based on the Griess method. The nitrite/nitrate levels in patients with degenerative lumbar disease (DLD), but not those with fracture or appendicitis, were significantly higher than those in an age-matched control group. The duration of pain in the DLD group was much longer than that in the fracture or appendicitis group. The nitrite/nitrate levels in the middle-aged and elderly DLD patients depended on the duration of pain. These data probably suggest that the duration of pain is critical for the elevation in nitrite/nitrate levels.

An electrochemical microprobe for detecting nitric oxide release in brain tissue.

To detect the release of nitric oxide (NO) in brain tissue, an electrochemical microprobe was developed. The output current of the probe correlated linearly with the NO concentration at the tip, and the sensitivity of the probe was between 3.5 and 106 pA per 1 microM change in NO concentration. This probe showed no sensitivity to oxygen or to oxidized derivatives of NO. The NO release from sodium nitroprusside solution was successfully detected by the probe. An NO probe inserted into the molecular layer of a rat cerebellar slice detected a response corresponding to 8-58 nM of NO concentration following electrical stimulation of the white matter. This response was blocked reversibly by tetrodotoxin (1 microM) and was attenuated in the medium containing hemoglobin (1 or 10 microM). The dependence of the response amplitude on the voltage at the cathode in the probe was the same as that of the NO-induced probe current. These results ensure that the NO probes developed in this study effectively detect the endogenous NO release in brain tissue.

Conditioned heart rate response: testing under anaesthesia in rats.

Classically conditioned heart rate response was studied in anaesthetized rats. Rats were conditioned with photic stimuli followed by electric tail shocks in a conscious state and tested under anaesthesia for conditioned heart rate change. Testing photic stimuli evoked a transient bradycardia in about one-third of testing trials. Magnitude of the bradycardia was significantly greater in conditioned rats than that in untrained or control rats trained with electric tail shocks followed by photic stimuli. The difference was found to be mainly due to the difference in amplitude of individual bradycardia response rather than that in frequency of occurrence of the response. Testing stimuli also evoked a slight and sustained tachycardia, but magnitude of the tachycardia in conditioned rats did not differ significantly from that in control or untrained rats. These results suggest that effects of classical conditioning on autonomic functions can be detected and studied under anaesthesia.

Noxious inputs to supraoptic neurosecretory cells in the rat.

The effects of noxious stimuli were studied on discharge activity of the neurosecretory cells identified in the supraoptic nucleus by antidromic excitation after pituitary stimulation, in anaesthetized rats. Tail pinching excited 24% and inhibited spontaneous discharge of 6% of the 91 cells tested. Noxious heat stimuli (44-63 degrees C) applied to the hindlimb paw produced a transient excitation in 26% of the 23 cells tested. Electric stimulation of either the sciatic or cutaneous nerve with 20-Hz pulses for 1 s, at an intensity 5 times stronger than the threshold for evoking the changes in respiratory movements and blood pressure similar to those after tail pinching or noxious heat stimuli, excited about 30% of the cells tested. The excitation produced by these noxious stimuli preceded, on some occasions, the respiratory movement and blood pressure decrease which occurred concomitantly. Peristimulus time histograms of spontaneous discharges constructed during stimulation of either nerve at 1 Hz, revealed the presence of excitatory synaptic inputs in about 35% of the neurosecretory cells tested. These data indicate the existence of direct neural pathways which mediate excitatory synaptic inputs originating from nociceptors to supraoptic neurosecretory cells. Since 9 of the 22 cells which were excited by tail pinching exhibited a "phasic" pattern of spontaneous discharge which is known to characterize certain vasopressin-secreting neurones in rats, it is suggested that these excited cells were, at least in part, vasopressinergic.

A quick test for sound discrimination ability of rats in a single session after preparatory training.

We developed a sound discrimination test combined with a preparatory training procedure. After water deprivation of 48 h, rats were trained to respond to a sound with pedal-pressing to receive a reward of water. Six of the eight trained rats showed pedal-pressing responses to the sound within 4 h of training, and these six were exposed to two different sounds, response to only one of which was rewarded with water. In a single session of 10 h, the rate and latency of pedal-pressing in response to the two sound stimuli were continuously monitored. All six tested rats showed behavioral discrimination between the rewarded and unrewarded sounds within 6 h.

Functional brain block preparation of the rat auditory cortex.

To maintain neural functions in brain block preparations of the rat auditory cortex in vitro, a pressurized oxygenated medium was injected into the blocks. Distribution of indigo carmine contained in the injection medium indicated that a columnar region of 1-2 mm in diameter was homogeneously perfused from the white matter to the pial surface. Stimulation of cortical layers just above the white matter produced supragranular field potentials of two negative peaks. They represented antidromic and postsynaptic activities, of which only the latter was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). The depth profile and temperature-dependency of field potentials in the blocks were very similar to those recorded in usual slice preparations. The responses in blocks were recorded stably for several hours. The functional brain block preparation may be a useful tool for analyses of neocortical neural networks in vitro.

Long-term potentiation of Ca2+ signal in the rat auditory cortex.

The Ca2+ signal in supragranular layers of the rat auditory cortex (AC) was studied in slice preparations using rhod-2, a Ca2+ indicator. White matter stimulation elicited an increase in the Ca2+ signal, which was maximal in the image taken 34 ms after stimulation. This peak time was the same as that of the Ca2+ signal in pyramidal neurons injected with rhod-2. The intensity of the Ca2+ signal was proportional to the amplitude of the field potentials in supragranular layers. The Ca2+ signal was inhibited almost completely by 200 microM Ni2+ , but only slightly by 50 microM D-2-amino-5-phosphonovalerate (APV), an NMDA-receptor antagonist. Tetanic stimulation of the white matter or supragranular layers elicited long-term potentiation (LTP) of the Ca2+ signal in AC slices, but the potentiation was not clear in slices of the visual cortex (VC). The induction of LTP of the field potentials in AC slices was blocked by 50 microM APV or 50 microM Ni2+. These results indicate that Ca2+ influx through Ni2+ -sensitive Ca2+ channels in pyramidal neurons is potentiated by tetanic stimulation in parallel with LTP of neural activities and might be important for the induction of LTP in AC slices.

Long-lasting enhancement of sound discrimination ability after sound exposure in rats.

Changes in the sound discrimination ability of rats were investigated after sound exposure (SE) in a Skinner box. For estimation of the sound discrimination ability, two different amplitude-modulated (AM) sounds (S+ and S-) were presented to the rats deprived of water for 48 h. Pedal press behavior in response to only S+ was rewarded with water. The percentages of trials in which pedal press behavior occurred in response to S+ or S- were calculated separately, and test performance of the rats was determined from the difference between the percentages. Rats were exposed to AM sounds during SE of 48 h, and the sound discrimination test was carried out. Enhancement of discrimination between S+ and S- was elicited by SE in a stimulus-specific manner. Latent extinction of the pedal press behavior in response to sound stimuli was not clearly found after SE. The enhancement of test performance was detected 1-48 h after the cessation of SE, and was blocked by injection of an antagonist of N-methyl-D-aspartate receptors into the auditory cortex bilaterally, immediately before the initiation of SE. These results suggest that SE elicits enhancement of sound discrimination ability, and the responsible site is in the auditory cortex.

Irreversible impairment of inhibitory neurons and nitric oxide release in the neocortex produced by low temperature and hypoxia in vitro.

Brain ischemia causes irreversible hyperexcitability, which may be attributed to irreversible impairment of inhibitory neurons. However, the conditions required for selective and irreversible impairment of inhibitory interneurons in vitro are unknown. In this study, we found that a combination of low temperature and hypoxia produced hyperexcitability in the neocortex. Neocortical tissue blocks isolated from rats were exposed to low temperature (1-3 degrees C) for 45 min and subsequently to room temperature (21-23 degrees C) for 60 min in the non-oxygenated medium. In experimental slices prepared from the processed blocks, hyperexcitability, similar to that elicited by an antagonist of GABA(A) receptors, was observed. Exposure of the neocortical tissue blocks to low temperature alone or room temperature alone did not elicit hyperexcitability. The excitability of pyramidal neurons, excitatory synaptic transmission and inhibitory effects of an agonist of GABA(A) receptors were normal in experimental slices. However, excitation of pyramidal neurons was inhibited after local stimulation of inhibitory neurons in control slices, but not in experimental slices. Nitric oxide (NO) release from cortical interneurons was also markedly reduced in experimental slices. These results indicate that irreversible impairment of neocortical inhibitory neurons was produced by low temperature combined with hypoxia produced in vitro.

Calcium-dependent and ouabain-resistant oxygen consumption in the rat neurohypophysis.

To analyze rapid changes in energy metabolism in the neurohypophysis, pO2 was measured in the tissue in vitro with a miniature O2 electrode (tip diameter less than 100 microns, 90% response time less than 3 s). Electrical stimulation (20 Hz, 5 s) evoked immediate pO2 decreases by 93.4 +/- 10.5 mm Hg (mean +/- S.E.M., n = 12) which lasted for about 1 min and were blocked by tetrodotoxin (1 microM) or sodium cyanide (1 mM). Replacement of Ca2+ in the perifusing medium with Mn2+ reduced the pO2 decreases to 23.1 +/- 4.9% (n = 5) of the value before the replacement. In normal medium, ouabain application (1 mM, 3 min) suppressed the electrically evoked pO2 decreases only slightly to 82.6 +/- 6.5% (n = 5). In the Mn2+ medium, the same ouabain application suppressed the pO2 changes to 28.8 +/- 1.4%. High K+ (70 mM) evoked pO2 decreases by 175.8 +/- 14.9 mm Hg (n = 5) within 1-2 min. These pO2 changes were reduced to 35.6 +/- 3.8% in an Mn2+ medium. Veratridine (100 microM) evoked pO2 decreases by 204.8 +/- 36.3 mm Hg (n = 5). During the pO2 decreases, the effects of electrical or high K+ stimulation on pO2 were blocked. These results indicate that O2 consumption was evoked by electrical stimulation, and probably that high K+ or veratridine application in the neurohypophysis is mainly dependent on extracellular calcium and resistant to ouabain. The relationship between O2 consumption and exocytotic release is discussed.

Synergistic interactions between footshocks and non-osmotic hypovolemia on vasopressin secretion in rats.

The effects of i.p. injected hypertonic NaCl and polyethylene glycol on the magnitude of increase in plasma vasopressin after footshocks were studied in male rats, to determine whether hypovolemia and body fluid osmolality interact with noxious stimuli on vasopressin secretion. Present data have demonstrated that non-osmotic hypovolemia but not body fluid hyperosmolarity interact significantly and synergistically with footshocks to potentiate vasopressin secretion.

Demonstration of reversible membrane internalization after exocytosis in the rat neurohypophysis.

Isolated rat neurohypophysis was stimulated electrically in media containing fluid phase markers such as carboxyfluorescein and choline. After the markers in the extracellular space were washed out, release of the markers trapped in the tissue was evoked by stimulation. Both the uptake and the release of fluid phase markers were not observed in a Mn2+-containing medium. These results provide direct evidence that internalized vesicles have the function to fuse with plasma membrane in response to Ca2+ entry during electrical stimulation.

Long-term synaptic changes in rat cerebellar slices reflected in extracellular K+ activity.

In rat cerebellar slices, a [K+]o increase evoked locally by molecular layer stimulation was recorded by a K(+)-sensitive microelectrode. This K+ response was reduced clearly during application of low-Ca2+ medium, kynurenate or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Sustained depression of the K+ response by about 20% was observed after simultaneously but not after alternately combined stimulation of the molecular layer and the white matter/granule cell layer. It is concluded from these results that long-term depression of parallel fiber-Purkinje cell transmission can be detected as depression of the K+ response.

Long-term potentiation in the auditory cortex of adult rats.

Long-term potentiation (LTP) in the auditory cortex was studied in slices obtained from adult rats. White matter stimulation produced field potentials in layers II/III, which were composed of two negative waves followed by a slow positivity. The second negative and third positive waves were blocked by a low Ca2+ (0.48 mM) medium or by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM), while the first negativity unchanged. D-2-amino-5-phosphonovalerate (D-AP5, 50 microM) showed no clear effect on the field potentials. Tetanic stimulation of the white matter produced potentiation in the second negativity, and the potentiation was maintained for at least 30 min. D-AP5 blocked this potentiation completely. These data indicate that LTP is evoked by activation of N-methyl-D-aspartate (NMDA) receptors in the auditory cortex of adult rats.

Effects of naloxone and of intraperitoneal hypertonic saline upon oxytocin release and upon supraoptic neuronal activity.

Urethane anaesthetized male rats were given an i.p. injection of hypertonic saline to increase plasma osmotic pressure. This injection resulted in significantly elevated plasma oxytocin levels and increased discharge activity of putative oxytocin cells in the supraoptic nucleus. Subsequent injection of naloxone (1 mg/kg) i.v. resulted in a similarly large increase in plasma oxytocin, but did not affect the discharge activity of putative oxytocin neurones. The results suggest that, following an i.p. injection of hypertonic saline, endogenous opioids act at the neurosecretory terminals to partially inhibit oxytocin release.

Eye movement-related activities in cells of the lateral suprasylvian cortex of the cat.

Investigation of eye movement-related activities and photic responsiveness using behaving cats demonstrated distinctive representations of eye movement signals in different areas of the lateral suprasylvian cortex: visual reafference in the medial bank of the middle suprasylvian sulcus and non-visual signals (proprioceptive reafference or efference copy) in the lateral bank.

Long-lasting memory of sounds combined with reward in rats.

We investigated the effects of sound stimuli combined with reward on the subsequent sound discrimination. Water-deprived rats were exposed to one of two sounds (S+ or S-) in a trial, and licking a spout only during the presentation of S+ was rewarded with water. The percentage of trials in which licking occurred was calculated separately for S+ and S-, and sound discrimination was estimated from the difference in the percentage. S+ and S- were significantly discriminated during an 8 h period. In the second test after 1-2 weeks, sound discrimination for the same S+ and S- was significantly better than that for the S+ of the previous S- and S- of the previous S+. These findings indicate that the memory of the sounds combined with reward in the first test was maintained for 1-2 weeks.