Development of the GABA system in organotypic culture of hippocampal and cerebellar slices from a 67-kDa isoform of glutamic acid decarboxylase (GAD67)-deficient mice

Mice lacking glutamic acid decarboxylase 67kDa (GAD67) are born with much reduced GABA contents but do not survive after birth. To estimate the subsequent development of GAD67-deficient neural tissue, the hippocampal and cerebellar slices were prepared while the mice were alive, and subjected to organotypic culture. Although GABA contents were initially less than 5%, they were markedly increased, and GABA-containing fiber networks were well developed during the culture, indicating that GAD65, the other GAD isoform, largely compensates for the lack of GAD67. Cerebellar Purkinje cells developed similarly in culture.     

Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain

The distribution of glutamic acid decarboxylase (GAD) mRNA was investigated throughout the rat brain by means of in situ hybridization. Hybridization was carried out with a 35S-radiolabeled cRNA probe transcribed from a cDNA from cat occipital cortex and cloned in a SP6-T7 promoter-containing vector. Fixed tissue sections were hybridized with 35S GAD probe (0.6 kb length). Signal was detected by means of film or emulsion autoradiography. The autoradiograms were semiquantitatively evaluated by means of computer-assisted image analysis. The results obtained with this evaluation were correlated with the results of the semiquantitative analysis of GAD immunoreactivity performed by Mugnaini and Oertel. Specific labeling was only observed in neuronal cell bodies, whereas no labeling was found over neuropil, glial and endothelial cells. The highest labeling was found in the bulbus olfactorius (internal plexiform and granular layers) and in the caudal magnocellular nucleus of the hypothalamus. Strong labeling was observed in the Purkinje layer of the cerebellar cortex, the interpeduncular nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch and the suprachiasmatic nucleus. Intermediate or low levels of GAD mRNA were present in various brain nuclei, where gamma-aminobutyric acid (GABA)-containing cell bodies had been observed with other techniques. Interestingly, a low level of GAD mRNA was found in the caudate-putamen and nucleus accumbens, where the vast majority of nerve cells is known to contain GAD immunoreactivity. Only a poor correlation was found between the present semiquantitative measurements of GAD mRNA content and previous analyses of the number of GAD-immunoreactive cell bodies. The present study demonstrates that there exists a differential regional expression of GAD mRNA. The comparison with cell counts performed by immunocytochemistry suggests that some brain areas, such as caudate-putamen and nucleus accumbens, contain a large number of GAD-immunoreactive cell bodies which express a low level of GAD mRNA. The opposite seems to be true for other nuclei, such as the globus pallidus, the zona reticulata of the substantia nigra and the inferior collicle, where few GAD-immunoreactive cell bodies contain high levels of GAD mRNA. In conclusion, the present study gives a low magnification map of GAD mRNA levels in the adult male rat brain. Marked biochemical heterogeneities may be present among GABA neuronal populations based on their expression of GAD mRNA. The comparison between the present in situ hybridization and previous immunocytochemical studies suggests that there may exist at least two populations of GABA neurons in the brain, having high and low levels respectively of both GAD mRNA and GAD enzyme.     

Efficient generation of reciprocal signals by inhibition

Reciprocal activity between populations of neurons has been widely observed in the brain and is essential for neuronal computation. The different mechanisms by which reciprocal neuronal activity is generated remain to be established. A common motif in neuronal circuits is the presence of afferents that provide excitation to one set of principal neurons and, via interneurons, inhibition to a second set of principal neurons. This circuitry can be the substrate for generation of reciprocal signals. Here we demonstrate that this equivalent circuit in the cerebellar cortex enables the reciprocal firing rates of Purkinje cells to be efficiently generated from a common set of mossy fiber inputs. The activity of a mossy fiber is relayed to Purkinje cells positioned immediately above it by excitatory granule cells. The firing rates of these Purkinje cells increase as a linear function of mossy fiber, and thus granule cell, activity. In addition to exciting Purkinje cells positioned immediately above it, the activity of a mossy fiber is relayed to laterally positioned Purkinje cells by a disynaptic granule cell → molecular layer interneuron pathway. Here we show in acutely prepared cerebellar slices that the input-output relationship of these laterally positioned Purkinje cells is linear and reciprocal to the first set. A similar linear input-output relationship between decreases in Purkinje cell firing and strength of stimulation of laterally positioned granule cells was also observed in vivo. Use of interneurons to generate reciprocal firing rates may be a common mechanism by which the brain generates reciprocal signals.     

Normalization of glutamate decarboxylase gene expression in the entopeduncular nucleus of rats with a unilateral 6-hydroxydopamine lesion correlates with increased GABAergic input following intermittent but not continuous levodopa

The expression of mRNA encoding for the 67 kilodalton isoform of glutamate decarboxylase (GAD67) was examined by in situ hybridization histochemistry in the entopeduncular nucleus (EP) of adult rats with a 6-hydroxydopamine unilaterally lesion of dopamine neurons. Our results provide original evidence that continuous or intermittent levodopa administration is equally effective at reversing the lesion-induced increase in GAD67 mRNA expression in the EP when compared with vehicle controls. To characterize the GABAergic interactions that may mediate levodopa-induced alterations in the EP, double-labeling in situ hybridization was conducted with a combination of GAD67 radioactive and preproenkephalin or preprotachykinin digoxigenin-labeled complementary RNA probes in the striatum. Levels of GAD67 mRNA labeling were significantly increased by intermittent, but not continuous levodopa. Analysis at the cellular level in a dorsal sector of the striatum revealed that GAD67 mRNA levels increased predominantly in preproenkephalin-unlabeled neuronal profiles, presumably striatal/EP neurons (+99.3%). Saturation analyses of (3)H-flunitrazepam binding to GABA(A) receptors in the EP showed that the increase in GAD67 mRNA in preproenkephalin-unlabeled neurons by intermittent levodopa paralleled a significant decrease in number of GABA(A) receptors (Bmax) in the EP ipsilateral to the lesion. Continuous levodopa failed to alter striatal GAD67 mRNA levels, or the number or affinity of GABA(A) receptors when compared with vehicle-treated controls. These results suggest the normalization of GAD gene expression in the EP by intermittent levodopa involves an increase in GABAergic inhibition by striatonigral/EP neurons of the direct pathway. Conversely, the effects of continuous levodopa on GAD mRNA levels in the EP do not appear to be mediated by GABA.     

Overall distribution of GLYT2 mRNA-containing versus GAD67 mRNA-containing neurons and colocalization of both mRNAs in midbrain, pons, and cerebellum in rats

We aimed to clarify the overall distribution of glycinergic neurons in the midbrain, pons, and cerebellum in rats, using in situ hybridization for mRNA encoding glycine transporter 2 (GLYT2), which reliably detects glycinergic cell bodies. We combined this method with in situ hybridization for mRNA encoding glutamic acid decarboxylase isoform 67 (GAD67), and have presented for the first time global and detailed views of the distribution of glycinergic neurons in relation to GABAergic neurons. In addition to this single-detection study, we performed double-detection of GLYT2 mRNA and GAD67 mRNA to determine the distribution of neurons co-expressing these mRNAs. We have shown that many areas of the brainstem and cerebellum, not only areas where previous immunohistochemical studies have specified, involve double-labeled neurons with GLYT2 and GAD67 mRNAs. In particular, when lightly labeled GLYT2 mRNA-positive neurons were distributed within the area of GAD67 mRNA-positive neurons, almost all such GLYT2 mRNA-positive neurons were GAD67 mRNA-positive. Areas or neuron groups expressing exclusively GLYT2 mRNA or GAD67 mRNA were rather limited, such as the superior colliculus, nucleus of the trapezoid body, and Purkinje cells. The present study suggests that the corelease of glycine and GABA from single neurons is more widespread than has been reported.     

Baro-activated neurons with pulse-modulated activity in the rat caudal ventrolateral medulla express GAD67 mRNA

GABAergic neurons in the caudal ventrolateral medulla (CVLM) are believed to mediate the sympathetic baroreceptor reflex by inhibiting presympathetic neurons in the rostral ventrolateral medulla (RVLM). Accordingly, some CVLM neurons are activated by increased arterial pressure (AP; baro-activated), have activity strongly modulated by the AP pulse (pulse-modulated), and can be antidromically activated from the RVLM. This study examined whether baro-activated, pulse-modulated CVLM neurons are indeed GABAergic and examined their structures. We recorded extracellularly from 19 baro-activated, pulse-modulated CVLM neurons in chloralose-anesthetized rats. Most of these cells (13/19) were silenced by decreasing AP with nitroprusside, but some (6/19) remained active at low AP levels. They were also excited by phenyl biguanide (17/17) but inhibited by noxious tail pinch (8/11). Twelve baro-activated cells were filled with biotinamide and examined for expression of GAD67 mRNA. Because adjacent vagal motor neurons are also activated by increased AP, we examined choline acetyltransferase (ChAT) immunoreactivity. Most baro-activated cells (9/12) expressed high levels of GAD67 mRNA, the rest (3/12) displayed lower levels of GAD67 mRNA, but none showed ChAT immunoreactivity. In contrast, adjacent baro-inhibited CVLM cells had no GAD67 mRNA (n = 5) but were instead tyrosine hydroxylase immunoreactive (n = 7). Reconstruction of baro-activated CVLM neurons revealed axons that projected dorsomedially and rostrally with several axon collaterals. These data demonstrate the existence of GABAergic CVLM neurons with the physiological characteristics expected of interneurons that mediate the sympathetic baroreceptor reflex. In addition, baro-activated GABAergic CVLM neurons appear to integrate several types of inputs and provide inhibition to multiple targets.     

Altered physiology of Purkinje neurons in cerebellar slices from transgenic mice with chronic central nervous system expression of interleukin-6

The cytokine interleukin-6 is produced at elevated levels within the central nervous system in a number of neurological diseases and has been proposed to contribute to the histopathologic, pathophysiologic, and cognitive deficits associated with such disorders. In order to determine the effects of chronic exposure of interleukin-6 on the physiology of central neurons, we compared the firing properties of cerebellar Purkinje neurons from control mice and transgenic mice that chronically express interleukin-6 within the central nervous system. Extracellular recordings from cerebellar slices revealed that the mean firing rate of spontaneously active Purkinje neurons was significantly reduced in slices from transgenic mice compared to control mice. In addition, a significantly greater proportion of Purkinje neurons from transgenic slices exhibited an oscillatory pattern of spontaneous firing than neurons in control slices. Orthodromic stimulation of climbing fiber afferents evoked similar excitatory synaptic responses (complex spikes) in Purkinje neurons of both transgenic and control mice. However, the inhibitory period following the complex spike (climbing fiber pause) was significantly longer in slices from transgenic mice. Using immunohistochemistry, we also showed that Purkinje neurons express high levels of both the interleukin-6 receptor and its intracellular signaling subunit, gp130, indicating that interleukin-6 could act directly on Purkinje neurons to alter their physiological properties. The interleukin-6 expressing transgenic mice have been shown previously to exhibit a number of histopathological changes in the central nervous system including injury and loss of cerebellar Purkinje neurons. The present data show that these transgenic mice also have altered physiology of cerebellar Purkinje neurons, potentially through a direct activation of interleukin-6 receptors expressed by this neuronal type. Interleukin-6 induced alterations of Purkinje neuron physiology would ultimately affect the flow of information out of the cerebellum, and could thus contribute to the motor deficits observed in the transgenic mice.     

Structural and functional alterations of cerebellum following fluid percussion injury in rats

Cerebellum was shown to be vulnerable to traumatic brain injury (TBI) in experimental animals. However, the detailed pathological and functional changes within the cerebellum following TBI are not known. Using our established cerebellum fluid percussion injury (FPI) model, we characterized the temporal pattern and the nature of structural damage following FPI, as well as the functional changes of Purkinje cells in response to climbing fiber activation. Our results showed that 60% of Purkinje cells died within the first 24 h following moderate FPI. In contrast, clusters of densely stained shrunken granule cells were stained positive for terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) in 1, 3 or 7 days following FPI animals. We also observed an accompanying structural damage to the cerebellar white matter tract. Disconnected axonal fibers appeared 1 day post-FPI, and loss of white matter fibers were visible 3 and 7 days post-FPI. Massive accumulation of β-amyloid precursor protein (βAPP) was found in the white matter tracts and molecular layer in the cerebellum of 1, 3 or 7 days FPI animals. Our functional study showed that the majority of Purkinje cells from 1 day and all cells from 3 to 7 days post-FPI had distorted membrane potential and synaptic responses to climbing fiber activation. These results suggested that there is a co-related structural and functional deterioration with a specific temporal pattern in the cerebellum following FPI. These observations provide a basis for future mechanistic investigations aiming to realize neuroprotection from cerebellar neuronal death and loss of cerebellar functionality.     

Influence of functional glia on the electrophysiology of Purkinje cells in organotypic cerebellar cultures

Previous studies have shown that exposure of organotypic cerebellar explants to cytosine arabinoside (Sigma) for the first five days in vitro drastically reduced the granule cell population and severely affected glial function. Myelination was absent and astrocytes failed to ensheath Purkinje cells. In the absence of astrocytic ensheathment, Purkinje cell somata became hyperinnervated by Purkinje cell recurrent axon collaterals. Recurrent axon collaterals also projected to Purkinje cell dendritic spines. In later studies, exposure of cerebellar cultures to a different formulation of cytosine arabinoside (Pfanstiehl) also affected granule cells and oligodendrocytes but did not compromise astrocyte function. The different susceptibility of astrocytes to the two preparations of cytosine arabinoside (Sigma and Pfanstiehl) has provided the opportunity to examine the electrophysiological properties of Purkinje cells in the presence and absence of functional glia. Ensheathed Purkinje cells in granuloprival cultures exhibit within two weeks in vitro similar passive membrane properties as Purkinje cells in control cultures. Their input resistance is significantly higher and their spontaneous single-unit discharge is significantly lower than that of unensheathed Purkinje cells. This effect suggests that ensheathed Purkinje cells in cytosine arabinoside (Pfanstiehl)-treated cultures are more responsive to the profuse Purkinje cell recurrent axon collateral inhibitory projection to dendritic spines. These studies also show that the presence of functional glia and/or astrocytic ensheathment can be correlated with the development of complex spike activity by Purkinje cells in vitro. Purkinje cells in cultures treated with cytosine arabinoside (Pfanstiehl), which does not compromise astrocytic ensheathment, display membrane conductances and spike activity similar to mature Purkinje cells in control cultures. By contrast, Purkinje cells in cultures treated with cytosine arabinoside (Sigma), and devoid of astrocytic ensheathment, display mainly simple spike activity reminiscent of the type of activity seen in less mature neurons.     

Maturation of evoked mossy fiber input to rat cerebellar Purkinje cells (II)

Summary The aim of this work was to establish a time sequence for the functional maturation of the mossy fiber afferent system to the cerebellum, in order to place it in the context of overall cerebellar cortical development. Rat pups at various ages were anesthetized with 0.5% halothane, and Purkinje cell activity was monitored extracellularly as limbs were electrically stimulated. The results showed that Purkinje cells can receive input from the periphery via the mossy fiber system at least by the seventh postnatal day, which is relatively early in overall cerebellar development. It is clear that synaptic transmission begins soon after the mossy fiber terminals and granule cell dendrites are in physical proximity. Initially, such input has a long latency (66 msec), is easily fatigued (at 10/sec), and has a prolonged duration of excitatory effect on Purkinje cells (27 msec). These and other functional parameters become mature by the third postnatal week (6–9 msec latency, following frequency above 20/sec), which is well before overall cerebellar cortical cytological development is completed. The maturation of many functional parameters of the cerebellar afferent systems and the acquisition of new motor behaviors are shown to emerge in parallel. Motor function appears to correlate with the establishment of a mature time scale of operation of the cerebellar circuitry.Supported by N. I. H. Grant 5-R01-GM00133 and N.S.F. Grant GB 43301     

NEURAL PLASTICITY IN CEREBELLAR CULTURES

Cerebellar granule cells and oligodendrocytes are destroyed and astrocytes are functionally compromised by exposure of organotypic cerebellar cultures derived from newborn mice to cytosine arabinoside for the first 5 days in vitro. Consequently, myelin does not form and Purkinje cells survive in increased numbers, but without astrocytic ensheathment. In the absence of glial sheaths, Purkinje cells have altered membrane properties and reduced input resistance. Their inhibitory recurrent axon collaterals sprout enormously and hyperinnervate the unensheathed somata of other Purkinje cells and form heterotypical synapses with Purkinje cell dendritic spines normally occupied by homotypical excitatory parallel fiber (granule cell axon) terminals. This reorganization of the cortical circuitry, in which recurrent axon collaterals are the dominant inhibitory elements, allows retention of some inhibition in the absence of parallel fiber excitation of the inhibitory interneurons. In the absence of neuronal activity, the full complement of inhibitory synapses is not developed and the cultures exhibit sustained cortical hyperactivity after recovery from the blockade. If granule cells and glia are replaced, a second round of reorganization ensues, in the direction of restoration of the normal cortical circuitry. The cultures are myelinated and the number of recurrent axon collaterals is reduced. Astrocytes ensheath Purkinje cell somata and strip excess axosomatic synapses, as well as eliminate some of the heterotypical synapses in the cortical neuropil. Parallel fibers synapse with already present Purkinje cell dendritic spines and with newly proliferated spines, the latter induced by an astrocyte secreted factor. As homotypical synapses develop and heterotypical synapses decline, Purkinje cells undergo apoptosis and their population is reduced to control levels. With the restoration of parallel fiber excitation, recurrent axon collaterals are no longer the dominant cortical inhibitory elements. If neuronal activity is blocked as the granule cells and glia are replaced, there is incomplete formation of inhibitory synapses, and cortical discharges are hyperactive after recovery from activity blockade. Copyright © 1996 Elsevier Science Ltd.     

Acute ethanol induces Fos in GABAergic and non-GABAergic forebrain neurons: a double-labeling study in the medial prefrontal cortex and extended amygdala

The purpose of this study was to further address the hypothesis that ethanol activates GABAergic neurons in specific brain neurocircuits that mediate motivated behavior and control of action, such as the central extended amygdala and medial prefrontal cortex. Male Sprague-Dawley rats received habituation to 7 days of daily intragastric administration of water (5 ml/kg) followed by a single acute intragastric dose of ethanol (2.5 g/kg) or water then, 2 h later, by paraformaldehyde perfusion. Rats left undisturbed in the animal room throughout the experiment were also perfused (naive group). Brain sections were processed for single Fos immunohistochemistry or dual Fos immunohistochemistry/glutamic acid decarboxylase (GAD) mRNA in situ hybridization. Intragastric water administration increased the number of Fos-immunoreactive cells in the infralimbic cortex and lateral part of the central nucleus of the amygdala compared with the naive group. Ethanol administration increased the number of Fos-immunoreactive cells in the infralimbic (+57.5%) and prelimbic (+105.3%) cortices, nucleus accumbens shell region (+88.2%), medial part of the central nucleus of the amygdala (+160%), and lateral part of the bed nucleus of the stria terminalis (+198.8%) compared with the water-treated group. In the nucleus accumbens shell region, central nucleus of the amygdala, and bed nucleus of the stria terminalis, more than 80% of Fos-immunoreactive neurons were GABAergic after ethanol administration. In contrast, in the prelimbic cortex, 75% of Fos-immunoreactive neurons were not GABAergic. These results constitute new evidence for region-specific functional interactions between ethanol and GABAergic neurons.     

Increased expression of GAD mRNA during the chronic epileptic syndrome due to intrahippocampal tetanus toxin

Summary A few mouse minimum lethal doses (MLD) of tetanus toxin injected into rat hippocampus triggers prolonged changes in neuronal function. Spontaneously recurring epileptic discharges arise in both the injected and the contralateral, uninjected hippocampus. The seizures remit after about 6 weeks, to be succeeded by a permanent depression of hippocampal neuronal responses. There is no evidence of any loss of pyramidal cells at this low dose of toxin. Here we studied presumptive inhibitory, GABAergic neurons, using in situ hybridization (ISH) with a probe directed against the mRNA encoding glutamic acid decarboxylase (GAD), at each of 1,2,4 and 8 weeks after injection of tetanus toxin. Epileptic activity was recorded from hippocampal slices prepared from both injected and contralateral hippocampi of rats at each time point, unexpectedly persisting until 8 weeks. There were no significant differences in the numbers of neurons containing GAD mRNA between toxin- and vehicle-injected and control rats in any hippocampal subfield, at any survival time, except for an apparently transient loss of hilar signal in vehicle-injected rats at 1 and 2 weeks which we attribute to a significant, transient loss of neuronal GAD mRNA to below the threshold for detection by ISH using this probe. In contrast there was a marked increase in GAD mRNA in the toxin-injected group, which reached a peak at 4 weeks, and returned to control levels by 8 weeks. The changes were bilateral and were most marked in the hilus of the dentate area, but were also significant in CA3 and CA1. Upregulation of GAD mRNA was preceded by an increase in the levels of the mRNA for the subunit of the GTP binding protein, Gs (Gs), at 2 weeks which affected the GABAergic neurons selectively, and not the pyramidal or granule cells. These marked changes in GAD mRNA may contribute to putative adaptive responses within GABAergic neurons, which would help contain epileptic activity in these chronic foci. The changes in GAD expression may be due to mechanisms acting through an increase in mRNA encoding Gs.     

GAD65-Reactive T cells in a Non-diabetic Stiff-man Syndrome Patient

GAD65 (glutamic acid decarboxylase) is an important autoantigen in both type 1 (insulin-dependent) diabetes mellitus (IDDM) and the neurological autoimmune disease stiff-man syndrome (SMS), and is expressed in pancreatic islets as well as the nervous system. Still, only 30% of SMS patients also have type 1 diabetes. To study regulation of T cell responsiveness to GAD65, we investigated a non-diabetic SMS patient with HLA-DR3/7 (predisposing to type 1 diabetes) and high levels of type 1 diabetes-associated autoantibodies against GAD65 and islet cells, and compared the results with those of her diabetic son and two other SMS patients. T cell responses to GAD65 were repeatedly absent in primary stimulation, whereas IA-2, islet antigen and tetanus toxoid induced significant T cell proliferation. However, after in vitro restimulation, GAD65 reactive T cell lines and clones were obtained that were HLA-DR3 restricted, and cross-reactive with a homogenate of purified human pancreatic islets. These T cells produced the immunoregulatory cytokine IL-10 in combination with IFN-gamma and IL-4 (Th0). The dominant T cell epitope was mapped to the central region of GAD65. Although no primary response to whole GAD65 was detectable, the naturally processed GAD65 peptide epitope was recognized vigorously in the primary stimulation assay. The lack of detectable primary T cell responses to GAD65, together with the GAD65-specific cytokine production of restimulated T cells, suggest that GAD65-specific cellular autoimmunity in this patient is suppressed and may be related to the absence of diabetes despite humoral autoreactivity and genetic predisposition.     

Streptozotocin upregulates GAD67 expression in MIN6N8a mouse beta cells

Glutamic acid decarboxylase (GAD) is one major autoantigen involved in the pathogenesis of autoimmune insulin dependent diabetes mellitus (IDDM). Molecular mechanisms regulating GAD expression in pancreatic beta cell are still ill-defined. Here we investigated the effect of streptozotocin (STZ), a beta cell-specific toxin, on the expression of GAD67 in MIN6N8a mouse beta cell. A 5-6-fold increase in the expression GAD67 mRNA was found in cells treated with 1.25mM STZ for 12h. Addition of NAD+ to the incubation medium slightly reduced the STZ-induced upregulation of GAD67. STZ increased p53 levels that in turn up-modulated GAD67 expression. This effect was abolished upon addition of the antioxidant N-acetyl cysteine (NAC). STZ also activated NF-kappaB and blockade of NF-kappaB activation inhibited the STZ-mediated upregulation of GAD67 expression. As a whole these data show that low dose of STZ up-regulates GAD67 expression in mouse bate cell and that NF-kappaB activation through oxidative stress plays a key role in this phenomenon. They also suggest that various stimuli promoting NF-kappaB activation may up-regulate expression of GAD autoantigen in mouse beta cells.     

Long-term effects of 3-acetylpyridine-induced destruction of cerebellar climbing fibers on Purkinje cell inhibition of vestibulospinal tract cells of the rat

Summary The inhibitory action of Purkinje cells on vestibulospinal tract (VST) cells was examined in rats deprived of climbing fibers with 3-acetylpyridine (3-AP) intoxication. In order to resolve discrepancies raised in previous studies with various means, special efforts were devoted to directly estimate Purkinje cell inhibition at synaptic levels by using intracellular recording, to avoid sampling bias by using a systematic survey of VST cells in each rat, and to evaluate the time-dependence of the effects of climbing fiber deafferentation by regular testing at 10 day intervals until 160 days after 3-AP intoxication. As compared with 661 VST cells impaled in 15 control rats, 1771 VST neurons impaled in 29 3-AP-treated rats revealed four basic changes in the monosynaptic inhibitory postsynaptic potentials (IPSPs) induced by stimulation of Purkinje cell axons in the white matter of the cerebellar anterior lobe. First, the rate of IPSP occurrence among VST cells was 0.64 in control rats; at more than 10 days after 3-AP intoxication it decreased gradually, down to 0.37–0.38 at the 70th–81st days, and thereafter increased up to 0.53 by the 160th day. The rate of IPSP occurrence varied considerably between the rostral and caudal regions, and also between the dorsal and ventral divisions of the VST cell population, but its reduction after 3-AP intoxication occurred approximately in parallel in all divisions. Second, IPSPs evoked with standard 500 A pulse stimuli were smaller in size on and after day 10. The reduction of IPSP size was by as much as 53% of control values at the 70th–101st days in the dorsal division, but no significant change occurred in the ventral division of the VST cell population. Third, the latency of the IPSPs was prolonged by about 0.25 ms on and after day 10. Analysis of the relationship between the IPSP latency and the dorsoventral location of VST cells in the medulla suggests that the major cause for the prolongation of IPSP latency is an increased synaptic delay at Purkinje cell axon terminals. Fourth, the cerebellar stimulation threshold for evoking IPSPs was almost always below 100 A in control rats, but values of 100–250 A were common after the 40th day. Thus, climbing fiber deafferentation exerts long-term influences on excitability of Purkinje cell axons, and on the connectivity and synaptic transmission from Purkinje cell axons to VST cells.On leave from the Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand     

Time-course of SKF-81297-induced increase in glutamic acid decarboxylase 65 and 67 mRNA levels in striatonigral neurons and decrease in GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, in adult rats with a unilateral 6-hydroxydopamine lesion

Striatal projection neurons use GABA as their neurotransmitter and express the rate-limiting synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter vGAT. The chronic systemic administration of an agonist of dopamine D1/D5-preferring receptors is known to alter GAD mRNA levels in striatonigral neurons in intact and dopamine-depleted rats. In the present study, the effects of a single or subchronic systemic administration of the dopamine D1/D5-preferring receptor agonist SKF-81297 on GAD65, GAD67, PPD and vGAT mRNA levels in the striatum and GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, were measured in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion. After a single injection of SKF-81297, striatal GAD65 mRNA levels were significantly increased at 3 but not 72 h. In contrast, striatal GAD67 mRNA levels were increased and nigral alpha1 mRNA levels were decreased at 72 but not 3 h. Single cell analysis on double-labeled sections indicated that increased GAD or vGAT mRNA levels after acute SKF-81297 occurred in striatonigral neurons identified by their lack of preproenkephalin expression. Subchronic SKF-81297 induced significant increases in striatal GAD67, GAD65, preprodynorphin and vGAT mRNA levels and decreases in nigral alpha1 mRNA levels. In the striatum contralateral to the 6-OHDA lesion, subchronic but not acute SKF-81297 induced a significant increase in GAD65 mRNA levels. The other mRNA levels were not significantly altered. Finally, striatal GAD67 mRNA levels were negatively correlated with nigral alpha1 mRNA levels in the dopamine-depleted but not dopamine-intact side. The results suggest that different signaling pathways are involved in the modulation by dopamine D1/D5 receptors of GAD65 and GAD67 mRNA levels in striatonigral neurons. They also suggest that the down-regulation of nigral GABA(A) receptors is linked to the increase in striatal GAD67 mRNA levels in the dopamine-depleted striatum.     

Sequential stimulation of rat and guinea pig cerebellar granular cells in vitro leads to increasing population activity in parallel fibers

Sequential stimulation of the granular layer of the cerebellar cortex in vitro using 11 linearly aligned stimulating electrodes leads to massive population activity in the parallel fiber system and to spike activity in Purkinje cells (Heck, D., Neurosci. Lett., 157 (1993) 95-98; Heck, D., Naturwissenschaften, 82 (1995) 201-2030). The induced parallel fiber activity, however, might have been a result of direct stimulation of parallel fibers themselves and not of stimulation of granular cells or their ascending axons. We report here that using sequential 'moving' stimuli and varying the distance covered by the 'movement', parallel fiber population spike amplitude increases with distance and saturates for distances longer than 1.0 mm. This effect cannot be explained if parallel fibers are directly stimulated, but requires stimulation of the granular cells or their ascending axons. We conclude that the population spike activity and Purkinje cell responses induced by sequential stimulation of the granular layer of the cerebellar cortex slices in this and earlier experiments consists of orthodromic parallel fiber spikes.