Tomás González-Hernández

Sources of GABAergic input to the inferior colliculus of the rat

We have studied the GABAergic projections to the inferior colliculus (IC) of the rat by combining the retrograde transport of horseradish peroxidase (HRP) and immunohistochemistry for γ‐amino butyric acid (GABA). Medium‐sized (0.06–0.14 μl) HRP injections were made in the ventral part of the central nucleus (CNIC), in the dorsal part of the CNIC, in the dorsal cortex (DCIC), and in the external cortex (ECIC) of the IC. Single HRP‐labeled and double (HRP‐GABA)‐labeled neurons were systematically counted in all brainstem auditory nuclei.

Compartmental organization and chemical profile of dopaminergic and GABAergic neurons in the substantia nigra of the rat.

The substantia nigra (SN) is a midbrain center composed of dopaminergic (DA‐) and gamma aminobutyric acid (GABA)ergic (GABA‐) neurons. In this study, we investigated the topographical relationship between both cell populations and their chemical profile by using single and double immunostaining for tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), cholecystokinin (CCK), calretinin (CR), calbindin (CB), parvalbumin (PV), and nitric oxide synthase (NOS). Our results showed that DA‐cells are arranged in two bands, one rostrodorsal that corresponds to the SN pars compacta (SNC), and another caudoventral that corresponds to the SN pars reticulata (SNR) and emits cell bridges that make contact with the rostrodorsal one. In the SNR, GABA‐cells are arranged in dorsoventrally elongated clusters that occupy DA‐cell free regions. According to cytoarchitectural, topographical, and chemical criteria, we identified ten different cell groups: five dopaminergic ones, and five GABAergic ones. Within DA‐cells, we found a cell group in the dorsomedial portion of the SNC which contains CCK, CR, and CB (dmSNC); DA‐cells in the SN pars lateralis (SNL) which also contain CCK, CR and CB; DA‐cells in the rostral half of the SNC containing CCK and CR (rSNC); DA‐cells in the SNR and the caudal half of the SNC which only express CR (cSNC‐SNR), and a DA‐cell group in the lateral part of the SNC that contains none of the markers studied (lSNC). Within GABA‐cells, we distinguished: large GABA‐cells in the SNL that contain PV; large GABA‐cells in the rostrolateral part of the SNR containing PV and NOS (rlSNR), small GABA‐cells in the caudomedial part of the SNR containing PV (cmSNR), and two groups of small GABA‐cells in the rostromedial portion of the SNR, one of them containing CR (rmcSNR), and the other containing NOS (rmnSNR). These data suggest that over a compartmental and complementary organization, DA‐ and GABA‐nigral cells form a mosaic of neurochemically different subnuclei which probably differ in their physiological and pharmacological properties and vulnerability to aggression. J. Comp. Neurol. 421:107–135, 2000.

Dopamine Cell Degeneration Induced by Intraventricular Administration of 6-Hydroxydopamine in the Rat: Similarities with Cell Loss in Parkinson's Disease

In an attempt to find a convenient rat model to study cell vulnerability in Parkinsons disease, we have investigated the cell-loss profile in different midbrain dopaminergic nuclei and subnuclei of rats injected with 6-hydroxydopamine (6-OHDA) in the third ventricle. Following administration of different doses (5-1000 microgram) of 6-OHDA, motor behavior was evaluated and tyrosine hydroxylase-immunostained neurons were counted in the A8 group and different subdivisions of A9 and A10 groups. Animals developed hypokinesia, repetitive chewing movements, and catalepsia. Signs of cell degeneration were evident from the first day after injection, reaching the definitive pattern at the end of the first week. There was a similar degeneration in both brain sides, the A9 group showing the highest degree of cell-loss, followed by A8 and A10 groups. In the A9 group, the degeneration mostly affected those subgroups located in its ventral, lateral, and posterior regions. In the A10 group the degeneration mainly affected the parabrachial pigmented nucleus, the paranigral nucleus and the ventral tegmental area. This topographic pattern of degeneration is very similar to that previously described in Parkinsons disease, suggesting that this model may be a useful tool in the study of the cell vulnerability mechanisms in this neurodegenerative disorder. In addition, our results also showed that small dopaminergic neurons are more resistant to degeneration than the large ones. In some DA subgroups, the cells that contained calbindin but not calretinin were less vulnerable to the neurotoxic effect of 6-OHDA.

Expression of dopamine and vesicular monoamine transporters and differential vulnerability of mesostriatal dopaminergic neurons.

Numerous studies suggest that the dopamine transporter (DAT), responsible for dopamine reuptake, may act as a vulnerability factor in the pathogenesis of Parkinsons disease (PD) and the vesicular monoamine transporter (VMAT2), responsible for its vesicular storage, as a neuroprotective factor. However, the relevance of each on the differential vulnerability of midbrain DA cells remains uknown. Here we studied the relationship between the expression pattern (mRNA and protein) of both transporters and the differential vulnerability of midbrain DA cells in a model of PD (intracerebroventricular injection of 6‐OHDA in rats) and in monkey and human midbrain. Our results revealed that the expression patterns for VMAT2 mRNA and protein and DAT mRNA are similar, with the highest levels in the rostromedial region of substantia nigra (SNrm), followed by the caudoventral region of SN (SNcv), the ventral tegmental area and pigmented parabrabraquial nucleus (VTA/PBP), and finally the linear and interfascicular nuclei (Li/IF). In contrast, the expression of DAT protein in rats, monkeys, and humans followed a caudoventrolateral‐to‐rostrodorsomedial decreasing gradient (SNcv > SNrm > VTA/PBP > Li/IF), matching the degeneration profile observed after intracerebroventricular injection of 6‐OHDA and in PD. In addition, DAT blockade made all midbrain DA cells equally resistant to 6‐OHDA. These data indicate that DAT protein levels, but not DAT mRNA levels, are closely related to the differential vulnerability of midbrain DA cells and that this relationship is unaffected by the relative levels of VMAT2. Furthermore, the difference between DAT mRNA and protein profiles suggests internuclear differences in its posttransductional regulation. J. Comp. Neurol. 479:198–215, 2004.

Consequences of unilateral nigrostriatal denervation on the thalamostriatal pathway in rats

The position of the caudal intralaminar nuclei within basal ganglia circuitry has largely been neglected in most studies dealing with basal ganglia function. During the past few years, there has been a growing body of evidence suggesting that the thalamic parafascicular nucleus in rodents (PF) exerts a multifaceted modulation of basal ganglia nuclei, at different levels. Our aim was to study the activity of the thalamostriatal pathway in rats with unilateral dopaminergic depletion. The experimental approach comprised first unilateral delivery of 6‐OHDA in the medial forebrain bundle. Thirty days post‐lesioning, animals showing a clear asymmetry were then subjected to bilateral injection of Fluoro‐Gold (FG) within the striatum. Subsequently, expression of the mRNA encoding the vesicular glutamate transporter 2 (vGLUT2) was detected within thalamostriatal‐projecting neurons (FG‐labeled) by in situ hybridization and the results were confirmed by laser‐guided capture microdissection microscopy followed by real‐time PCR. The data showed that there was a marked neuronal loss restricted to PF neurons projecting to the dopamine‐depleted striatum. Moreover, PF neurons innervating the dopamine‐depleted striatum were intensely hyperactive. These neurons showed a marked increase on the expression of vGLUT2 mRNA as well as for the mRNA encoding the subunit I of cytochrome oxidase as compared with those neurons projecting to the striatum with normal dopamine content. Thus, the selective neurodegeneration of PF neurons innervating the striatum together with the increased activity of the thalamostriatal pathway coexist after nigrostriatal denervation.

Motor behavioural changes after intracerebroventricular injection of 6-hydroxydopamine in the rat: an animal model of Parkinson's disease

At the beginning of the 1970s, different studies reported behavioural disturbances after the intracerebroventricular (icv) administration of 6-hydroxydopamine (6-OHDA) in the rat. Despite the fact that this neurotoxic agent degenerates brain dopaminergic (DA-) cells, its potential utility to produce a rat model of Parkinsons disease (PD) was never systematically studied because the aphagia and adipsia were often observed. In the present study, a procedure that induces a marked DA-cell degeneration that bypasses these and other undesirable complications of icv injection of 6-OHDA is reported. Lesioned animals (50-500 microg of 6-OHDA) showed a persistent motor syndrome composed of hypokinesia, purposeless chewing and catalepsy. The intensity of motor signs was dose-dependent, and recovered partially after administration of DA-receptor agonists, exposure to sensorial stimuli and stress, three procedures that reduce motor dysfunctions in Parkinsons disease (PD). Lesioned animals showed bilateral and symmetrical midbrain DA-cell degeneration with the highest cell-loss in A9 group (substantia nigra), followed by A8 (retrorubral field) and A10 (ventral tegmental area) groups. The similarity between the behavioural syndrome and the topographical profile of cell-loss after icv injection of 6-OHDA in rats and the clinical and neuropathological features of PD indicates that this may be a convenient animal model of PD particularly useful for checking in rats the possible efficacy of new anti-parkinsonian drugs on specific parameters of motor dysfunctions.

Expression of three forms of nitric oxide synthase in peripheral nerve regeneration

Nitric oxide (NO) is a short‐lived molecule with messenger and cytotoxic functions in nervous, cardiovascular, and immune systems. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis, exists in three different forms: the neuronal (nNOS), present in discrete neuronal populations; the endothelial (eNOS), present in vascular endotheliun, and the inducible isoform (iNOS), expressed in various cell types when activated, including macrophages and glial cells. In this study, we have investigated the possible involvement of NO in Wallerian degeneration and the subsequent regeneration occurring after sciatic nerve ligature, using histochemistry and immunocytochemistry for the three NOS isoforms, at different postinjury periods. Two days after lesion, the three NOS isoforms are overexpressed, reaching their greatest expression during the second week. nNOS is upregulated in dorsal root ganglion neurons, centrifugally transported and accumulated in growing axons. eNOS is overexpressed in vasa nervorum of the distal stump and around ligature, and iNOS is induced in recruited macrophages. These findings indicate that different cellular sources contribute to maintain high levels of NO at the lesion site. The parallelism between NOS inductions and well‐known repair phenomena suggests that NO, acting in different ways, may exert a beneficial effect on nerve regeneration. J. Neurosci. Res. 55:198–207, 1999. 

Striatal expression of GDNF and differential vulnerability of midbrain dopaminergic cells.

Glial cell line‐derived neurotrophic factor (GDNF) is a member of the transforming growth factor‐β superfamily that when exogenously administrated exerts a potent trophic action on dopaminergic (DA) cells. Although we know a lot about its signalling mechanisms and pharmacological effects, physiological actions of GDNF on the adult brain remain unclear. Here, we have used morphological and molecular techniques, and an experimental model of Parkinsons disease in rats, to investigate whether GDNF constitutively expressed in the adult mesostriatal system plays a neuroprotective role on midbrain DA cells. We found that although all midbrain DA cells express both receptor components of GDNF (GFRα1 and Ret), those in the ventral tegmental area (VTA) and rostromedial substantia nigra (SNrm) also contain GDNF but not GDNFmRNA. The levels of GDNFmRNA are significantly higher in the ventral striatum (vSt), the target region of VTA and SNrm cells, than in the dorsal striatum (dSt), the target region of DA cells in the caudoventral substantia nigra (SNcv). After fluoro‐gold injection in striatum, VTA and SNrm DA cells show triple labelling for tyrosine hydroxylase, GDNF and fluoro‐gold, and after colchicine injection in the lateral ventricle, they become GDNF‐immunonegative, suggesting that GDNF in DA somata comes from their striatal target. As DA cells in VTA and SNrm are more resistant than those in SNcv to intracerebroventricular injection of 6‐OHDA, as occurs in Parkinsons disease, we can suggest that the fact that they project to vSt, where GDNF expression is significantly higher than in the dSt, is a neuroprotective factor involved in the differential vulnerability of midbrain DA neurons.

Electrophysiological and Morphological Evidence for a GABAergic Nigrostriatal Pathway

The electrophysiological and neurochemical characteristics of the nondopaminergic nigrostriatal (NO-DA) cells and their functional response to the degeneration of dopaminergic nigrostriatal (DA) cells were studied. Three different criteria were used to identify NO-DA cells: (1) antidromic response to striatal stimulation with an electrophysiological behavior (firing rate, interspike interval variability, and conduction velocity) different from that of DA cells; (2) retrograde labeling after striatal injection of HRP but showing immunonegativity for DA cell markers (tyrosine hydroxylase, calretinin, calbindin-D28k, and cholecystokinin); and (3) resistance to neurotoxic effect of 6-hydroxydomine (6-OHDA). Our results showed that under normal conditions, 5–8% of nigrostriatal neurons are immunoreactive for GABA, glutamic acid decarboxylase, and parvalbumin, markers of GABAergic neurons, a percentage that reached 81–84% after 6-OHDA injection. Electrophysiologically, NO-DA cells showed a behavior similar to that found in other nigral GABAergic (nigrothalamic) cells. In addition, the 6-OHDA degeneration of DA cells induced a modification of their electrophysiological pattern similar to that found in GABAergic nigrothalamic neurons. Taken together, the present data indicate the existence of a small GABAergic nigrostriatal pathway and suggest their involvement in the pathophysiology of Parkinson’s disease.

Histochemical and immunohistochemical detection of neurons that produce nitric oxide: effect of different fixative parameters and immunoreactivity against non-neuronal NOS antisera.

This study focused on two points concerning the histochemical and immunohistochemical detection of neurons that produce nitric oxide (NO): (a) the effect of fixation and other methodological parameters on the staining pattern of both NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, and (b) the possibility that neurons display immunoreactivity against NOS antisera obtained from non-neuronal sources. Frontal sections of rat brains, fixed with 4% paraformaldehyde according to different protocols, were processed for single and double labeling using NADPH-d histochemistry and neuronal (nNOS), macrophagic (macNOS), and endothelial (eNOS) NOS immunohistochemistry. Our results show that variations in the fixative schedule, even within standard parameters, produce qualitative and quantitative changes in NADPH-d labeling. The effect of fixative on weakly stained neurons is different from that on heavily stained neurons. In subfixed brains, a large number of NOS-positive neurons lose their NADPH-d activity, whereas NOS immunolabeling remains unaltered. This finding may be particularly interesting in morphological studies that compare NADPH-d activity under experimental conditions that can affect brain perfusion. On the other hand, many cortical and subcortical neurons show macNOS immunoreactivity, most of it colocalized with nNOS.