Unlesioned Animals Research Articles

Chronic oral nicotine normalizes dopaminergic function and synaptic plasticity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned primates.

Our recent studies show that chronic oral nicotine partially protects against striatal damage in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates. To identify the cellular changes associated with this protective action, we investigated the effects of nicotine treatment on stimulus-evoked dopamine release, dopamine turnover, and synaptic plasticity in striatum from lesioned and unlesioned animals. Monkeys were chronically (6 months) treated with nicotine in the drinking water and subsequently lesioned with the dopaminergic neurotoxin MPTP (6 months) while nicotine was continued. Nigrostriatal damage increased nicotinic acetylcholine receptor (nAChR)-mediated fractional dopamine release from residual terminals, primarily through changes in alpha3*/alpha6* nAChRs. In contrast, fractional receptor-evoked dopamine release was similar to control in unlesioned and lesioned animals with chronic oral nicotine. Long-term nicotine administration also attenuated the enhanced K(+)-evoked fractional dopamine release from synaptosomes of MPTP-lesioned animals, suggesting that nicotine treatment had a generalized effect on dopaminergic function. This premise was further supported by experiments showing that nicotine dosing decreased the elevated dopamine turnover that occurs after nigrostriatal damage. We next investigated changes in synaptic plasticity with lesioning and nicotine treatment. Nicotine treatment alone enhanced synaptic plasticity by lowering the threshold for long-term depression (LTD) in the corticostriatal pathway. MPTP lesioning led to a loss of LTD, a measure of short-term synaptic plasticity. In contrast, LTD was preserved in nicotine-treated lesioned animals. Thus, the present data show that the disruptions in striatal dopaminergic function after nigrostriatal damage were attenuated with chronic nicotine administration. These cellular alterations may underlie the ability of nicotine to maintain/restore normal function with nigrostriatal damage.

BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats

Although regeneration of injured axons is inhibited within the adult CNS, moderate recovery can be found in patients and animals with incomplete spinal cord injury (SCI). This can be partly attributed to sprouting of spared and injured axons, rostral and caudal to the lesion, respectively. Recently, it has been reported that following a thoracic SCI such sprouting can result in indirect reconnections of the lesioned axons to caudal targets via propriospinal interneurons (PrI). Here, we attempted to further promote this spontaneous repair mechanism by applying the neurotrophic factor BDNF (brain-derived neurotrophic factor), in the vicinity of the cell bodies of lesioned corticospinal neurons or NT-3, intrathecally to the cervical spinal cord. We performed a dorsal over-hemisection at the thoracic spinal cord sparing only the left ventrolateral quadrant. This type of lesion did not promote sprouting of injured corticospinal axons or re-routing via commissural PrI. Also, in rats that received NT-3 at the cervical enlargement, no increase in sprouting was found. However, animals receiving BDNF at the cell bodies of lesioned corticospinal neurons showed a significant increase in collateral sprouting and in the number of contacts with PrI. This was not observed when BDNF was administered to unlesioned animals. Although no statistical difference in the horizontal ladder walking was found between the groups, the increase in collateral sprouting and in the number of contacts correlated with the functional recovery. Hence, cell body treatment can promote plasticity of the injured CNS and may be a valuable treatment approach in conjunction with local regeneration promoting strategies.

451. AAV-BDNF Augments Neurogenesis in Both the Normal Adult Rat Brain and the Quinolinic Acid Lesion Model of Huntington's Disease

The presence of ongoing neurogenesis in the adult mammalian brain raises the exciting possibility that endogenous progenitor cells could be used therapeutically for repair of neuronal loss associated with brain disease or injury. While previous studies have demonstrated that the adult brain has the potential for self-repair, it is apparent that the level of neurogenesis observed is insufficient to compensate for the progressive cell loss that occurs during brain injury and disease. In order for endogenous progenitor cells to be useful therapeutically, methods need to be developed to augment the process of neurogenesis and direct progenitor cells to proliferate, migrate and generate new neurons in specific areas of neuronal cell loss. Using in vivo gene transfer, we investigated the effect of Brain-Derived Neurotrophic Factor (BDNF) on the migration, differentiation and survival of endogenous progenitor cells in both the normal adult rat brain and the quinolinic acid (QA) lesion rat model of Huntington's disease (HD). We have developed a recombinant AAV vector encoding the human BDNF transgene under the control of the CBA constitutive promoter. Adult rats received unilateral stereotaxic injections of either AAV-BDNF, the control vector AAV-luciferase or sterile PBS into the anterior subventricular zone (SVZ). Three weeks later, the rats received daily injections of the mitotic marker bromodeoxyuridine (BrdU; 150 mg/kg i.p.) for 21 days in order to identify newly generated cells. Half of the rats also received a unilateral striatal injection of QA (50 nmol) in order to model HD. In the normal adult rat brain, BDNF over-expression had no effect on the number of BrdU labelled cells in the SVZ or striatum but resulted in a significant increase in BrdU labelling in the rostral migratory stream (RMS) compared to untreated animals (p|[le]|0.01). In untreated animals that received a striatal QA lesion we observed a significant reduction in the number of BrdU labelled cells in the SVZ and RMS compared to unlesioned animals (p|[le]|0.05) 21 days after lesioning, suggesting a chronic alteration in the dynamics of progenitor cell proliferation in response to QA lesioning. While BDNF over-expression did not alter the reduction in BrdU labelling observed in the RMS of QA lesioned animals, BDNF significantly increased the level of BrdU labelling in the SVZ of QA lesioned animals to that observed in unlesioned animals (p|[le]|0.01). BDNF also greatly induced the level of BrdU-immunoreactivity observed in the QA lesioned striatum compared to untreated animals (p|[le]|0.001). Furthermore, 9 weeks following QA lesioning we observed that BDNF over-expression had greatly enhanced the level of striatal neurogenesis observed in QA lesioned animals compared to untreated animals. These results demonstrate that over-expression of BDNF in the SVZ substantially augments the survival, migration and neuronal differentiation of progenitor cells in both neurogenic and non-neurogenic regions of the normal or QA lesioned adult rat brain, supporting the potential therapeutic use of endogenous neural progenitor cells for the treatment of HD.

Septal innervation regulates the function of α7 nicotinic receptors in CA1 hippocampal interneurons

The hippocampus receives substantial input from the medial septum/diagonal band of broca (MS/DB) via the fibria-fornix (FF). Projections from the MS/DB innervate hippocampal interneurons that express α7 nicotinic receptors and regulate excitation in principal cell populations. In the present report we used stereotaxic surgery, whole-cell patch clamping, and immunohistochemical techniques to evaluate the effects of FF and MS/DB lesions on α7 nicotinic receptors in stratum radiatum interneurons. Focal somatic application of ACh (1 mM) evoked methyllycaconitine (MLA)-sensitive currents that were markedly reduced following aspirative lesions of the FF. Reductions in current amplitudes were prevented or restored to levels not significantly different from controls following in vivo treatment with the α7-selective agonist GTS-21, and GTS-21 treatment did not change current amplitudes measured in tissue from unlesioned animals. MS/DB injections of the selective cholinergic neurotoxin 192 IgG-saporin did not affect α7 receptor currents, although MS/DB ChAT and hippocampal AChE immunolabeling were significantly reduced. In contrast, kainic acid lesions of the MS/DB, potentially more selective for GABAergic projection neurons, produced significant reductions in current amplitudes. These findings are the first to show functional changes in α7 receptors following hippocampal denervation and suggest that MS/DB hippocampal innervation regulates functional aspects of hippocampal α7 receptors. The results confirm hippocampal α7 nicotinic receptors as viable therapeutic targets in diseases that involve degradation of the septohippocampal pathway and may indicate that GABAergic MS/DB hippocampal input plays a more substantial role in the regulation of α7 nicotinic receptor function than MS/DB hippocampal cholinergic input.

3,4-Methylenedioxymethamphetamine and naloxone in rat rotational behaviour and open field

It has recently been shown that 3,4-Methylenedioxymethamphetamine (MDMA) has an anti-parkinsonian effect in rodent models of Parkinson's disease. The mechanism of this anti-parkinsonian action is unknown. Opioids have been suggested to play a role in MDMA-induced behaviour. We therefore investigated MDMA and naloxone in the rat rotational behavioural model. Male Sprague–Dawley rats were lesioned unilaterally with 6-hydroxydopamine at the medial forebrain bundle. Administration of R/S-MDMA (5 mg/kg, s.c.) produced ipsilateral rotations. Naloxone (2, 5, 10 mg/kg, s.c.) did not produce rotations on its own but reduced the number of MDMA-induced ipsilateral rotations. This effect was not dose-dependent. In contrast to reports on mice, in unlesioned animals, naloxone (10 mg/kg, s.c.) did not block MDMA (5 mg/kg, s.c.)-induced hyperactivity in an open field in our experiment. It is concluded that endogenous opioids play a role in MDMA's action in the rat rotational behavioural model.

Enhanced striatal opioid receptor-mediated G-protein activation in l-dopa-treated dyskinetic monkeys

Long-term l-3,4-dihydroxyphenylalanine ( l-dopa) treatment in Parkinson’s disease leads to dyskinesias in the majority of patients. The underlying molecular mechanisms for l-dopa-induced dyskinesias (LIDs) are currently unclear. However, the findings that there are alterations in opioid peptide mRNA and protein expression and that opioid ligands modulate dyskinesias suggest that the opioid system may be involved. To further understand its role in dyskinesias, we mapped opioid receptor-stimulated G-protein activation using [ 35S]guanylyl-5′- O-(γ-thio)-triphosphate ([ 35S]GTPγS) autoradiography in the basal ganglia of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned squirrel monkeys administered water or l-dopa. Subtype-selective opioid receptor G-protein coupling was investigated using the μ-opioid agonist [ d-Ala, N-Me-Phe, Gly-ol]-enkephalin, δ-agonist SNC80 and κ-agonist U50488H. Our data show that μ-opioid receptor-mediated G-protein activation is significantly enhanced in the basal ganglia and cortex of l-dopa-treated dyskinetic monkeys, whereas δ- and κ-receptor-induced increases were limited to only a few regions. A similar pattern of enhancement was observed in both MPTP-lesioned and unlesioned animals with LIDs suggesting the effect was not simply due to a compromised nigrostriatal system. Opioid receptor G-protein coupling was not enhanced in non-dyskinetic l-dopa-treated animals, or lesioned monkeys not given l-dopa. The increases in opioid-stimulated [ 35S]GTPγS binding are directly correlated with dyskinesias. The present data demonstrate an enhanced subtype-selective opioid-receptor G-protein coupling in the basal ganglia of monkeys with LIDs. The positive correlation with LIDs suggests this may represent an intracellular signaling mechanism underlying these movement abnormalities.

3,4‐Methylenedioxymethamphetamine counteracts akinesia enantioselectively in rat rotational behavior and catalepsy

We have shown recently that 3,4-methylenedioxymethamphetamine (MDMA) has symptomatic antiparkinsonian activity in rodent models of Parkinson's disease. In search of its mechanism of action, we further investigated the enantiomers of MDMA in the rotational behavioral model and catalepsy test. Catalepsy testing was done in drug-naive unlesioned animals. The parkinsonian symptoms rigor and akinesia (i.e., catalepsy) were induced by intraperitoneal administration of haloperidol 0.5 mg/kg and measured repeatedly as descent latency from a horizontal bar and a vertical grid. MDMA and both its enantiomers were effective in counteracting haloperidol-induced catalepsy, but if given as racemic, the effects were more pronounced than with the enantiomers. For testing of rotational behavior, male Sprague Dawley rats were lesioned unilaterally with 6-hydroxydopamine (6-OHDA) at the medial forebrain bundle. Administration of S-MDMA (5 mg/kg) produced ipsilateral rotations. R-MDMA was far less effective in inducing ipsilateral rotations in 6-OHDA unilaterally lesioned rats, but when S-MDMA was given additionally rotations immediately increased. Regarding their overall antiparkinsonian effects, the S-enantiomer of MDMA was more effective than its R-congener. R-MDMA was able to increase the actions of S-MDMA.

Conditioned suppression and freezing as measures of aversive Pavlovian conditioning: effects of discrete amygdala lesions and overtraining

Freezing and suppression are measures of conditioned fear that correlate in unlesioned animals. Both the basolateral (BLA) and central (CeN) nuclei of the amygdala are required for conditioned freezing, though there can be recovery with overtraining. The neuroanatomical substrates of conditioned suppression are less clear, with evidence both for a specific requirement of the CeN and for disruption by BLA lesions. The present study investigated the impact of selective excitotoxic lesions of the BLA and CeN upon the acquisition and expression of conditioned fear, measured by freezing and both on-baseline and off-baseline conditioned suppression in the same rats. BLA and CeN lesions both abolished all measures of conditioned fear after 9 trials of fear conditioning. However, when conditioning was extended to 33 trials, whereas rats with combined lesions of both the BLA and CeN continued to show no conditioned fear responses, there was a pattern of recovery observed after selective lesions. There was a partial recovery of freezing with both lesions, and full recovery of conditioned suppression, except for off-baseline suppression in CeN lesioned rats. These results indicate that with few conditioning trials, both the BLA and CeN are required in a serial manner for conditioned fear responses, but that overtraining can mitigate such impairments, likely involving parallel pathways in and through the amygdala.

Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression

Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes ( n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.

Tenascin-R as a repellent guidance molecule for newly growing and regenerating optic axons in adult zebrafish

In adult fish, in contrast to mammals, new optic axons are continuously added to the optic projection, and optic axons regrow after injury. Thus, pathfinding of optic axons during development, adult growth, and adult regeneration may rely on the same guidance cues. We have shown that tenascin-R, a component of the extracellular matrix, borders the optic pathway in developing zebrafish and acts as a repellent guidance molecule for optic axons. Here we analyze tenascin-R expression patterns along the unlesioned and lesioned optic pathway of adult zebrafish and test the influence of tenascin-R on growing optic axons of adult fish in vitro. Within intraretinal fascicles of optic axons and in the optic nerve, newly added optic axons grow in a tenascin-R immunonegative pathway, which is bordered by tenascin-R immunoreactivity. In the brain, tenascin-R expression domains in the ventral diencephalon, in non-retinorecipient pretectal nuclei and in some tectal layers closely border the optic pathway in unlesioned animals and during axon regrowth. We mimicked these boundary situations with a sharp substrate border of tenascin-R in vitro. Optic axons emanating from adult retinal explants were repelled by tenascin-R substrate borders. This is consistent with a function of tenascin-R as a repellent guidance molecule in boundaries for adult optic axons. Thus, tenascin-R may guide newly added and regenerating optic axons by a contact-repellent mechanism in the optic pathway of adult fish.

L-DOPA treatment modulates nicotinic receptors in monkey striatum.

Nicotinic acetylcholine receptor (nAChR) activation is well known to stimulate dopamine release in the striatum. This phenomenon may be physiologically significant in the control of motor function, as well as in pathological conditions such as Parkinson's disease. An understanding of the mechanisms that influence nAChR expression and function is therefore important. Because the dopamine precursor l-DOPA is the most commonly used therapeutic agent for Parkinson's disease, we investigated the effects of l-DOPA treatment on striatal nAChR expression in unlesioned and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. In unlesioned animals, l-DOPA (15 mg/kg) administered twice daily for 2 weeks decreased both 125I-epibatidine and [125I]iodo-3-[2(S)-azetidinylmethoxy]pyridine (A-85380) binding sites in the caudate and putamen, but did not affect 125I-alpha-CtxMII sites. alpha-CtxMII inhibition of striatal 125I-epibatidine and [125I]A-85380 binding with alpha-CtxMII suggest that there are both high- (Ki < 0.2 nM) and low-affinity (Ki > 100 nM) alpha-CtxMII-sensitive sites, as well as alpha-CtxMII-resistant sites, and that l-DOPA treatment influences only the low-affinity alpha-CtxMII-sensitive subtype. The l-DOPA effect was selective for striatal nAChRs with no change in cortical sites. Monkeys with severe nigrostriatal damage did not exhibit l-DOPA-induced declines in striatal nAChRs, suggesting that l-DOPA primarily affects nAChRs associated with dopaminergic terminals. In summary, these data show that l-DOPA treatment decreases nAChR expression, in contrast with the well established up-regulation of these sites by chronic nicotine exposure. Furthermore, they demonstrate preferential l-DOPA regulation of a novel low-affinity alpha-CtxMII-sensitive site. These declines in nAChRs with l-DOPA may be relevant to both the therapeutic and side effect profiles of l-DOPA therapy in Parkinson's disease.

Cerebrospinal fluid 3,4-dihydroxyphenylacetic acid level after tolcapone administration as an indicator of nigrostriatal degeneration

The development of reliable biological markers of nigrostriatal degeneration has important implications from both experimental and clinical viewpoints, since such biomarkers could be used for diagnostic and monitoring purposes in models of parkinsonism as well as in Parkinson’s disease patients. In this study, levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in the cerebrospinal fluid (CSF) of normal and parkinsonian squirrel monkeys in order to assess their reliability as indicators of nigrostriatal injury. In particular, we tested the hypothesis that these measurements may become more accurate by inhibiting catecholamine-O-methyltransferase (COMT) activity and therefore blocking the conversion of DOPAC to homovanillic acid. Oral administration of the COMT inhibitor tolcapone (2 doses of 15 mg/kg each with a 4-h interval) significantly reduced enzyme activity in the monkey brain. Tolcapone treatment enhanced CSF DOPAC concentrations in unlesioned animals (by approximately four times) as well as monkeys rendered parkinsonian after severe nigrostriatal dopaminergic injury caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, however, COMT inhibition greatly magnified the differences in CSF DOPAC levels between control and parkinsonian monkeys, since MPTP-induced DOPAC depletion was 35% in the absence vs >60% in the presence of tolcapone. Thus, tolcapone administration enhances the detection of DOPAC in the CSF and, by doing so, improves the reliability of CSF DOPAC as a marker of nigrostriatal degeneration.

Increases in striatal preproenkephalin gene expression are associated with nigrostriatal damage but not L-DOPA-induced dyskinesias in the squirrel monkey

Changes in preproenkephalin expression in the caudate and putamen have been linked to the development of L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias in primate models of Parkinson’s disease, although not all investigators have been able to confirm this association. Because nigrostriatal damage per se is associated with increases in striatal preproenkephalin mRNA levels, it is difficult to know if changes in transcript levels are a result of lesioning or concurrent L-DOPA treatment and resulting dyskinesias. To circumvent these difficulties, we measured striatal preproenkephalin mRNA levels in monkeys with L-DOPA-induced dyskinesias both with and without lesions of the nigrostriatal system. The latter model is not confounded by morphological and biochemical changes resulting from nigrostriatal damage. Monkeys were gavaged with L-DOPA (15 mg/kg) twice daily for a 2-week period and killed 3 days after treatment. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment alone resulted in an increase in preproenkephalin mRNA levels as previously shown. However, striatal transcript levels were similarly elevated in dyskinetic MPTP-lesioned animals treated with L-DOPA. In unlesioned animals, preproenkephalin mRNA levels were also similar in control and L-DOPA-treated dyskinetic monkeys. Because drug-induced changes in mRNA may not be sustained for a prolonged period after treatment, a second series of experiments were done in which animals were killed 3–4 h after the last dose of L-DOPA, but the results were similar to those obtained after 3 days. These data show that, while elevations in striatal preproenkephalin mRNA levels are associated with nigrostriatal damage, they are not linked to the development of L-DOPA-induced dyskinesias. These results thus question the importance of preproenkephalin mRNA in the pathogenesis of this disabling complication of L-DOPA therapy in Parkinson’s disease.

Increase in descending brain-spinal cord projections with age in larval lamprey: implications for spinal cord injury.

The purpose of this study was to determine whether new descending brain-spinal cord projections are added with age in larval lamprey and might contribute substantially to restoration of these projections following spinal cord injury. Retrograde horseradish peroxidase (HRP) labeling of descending brain neurons was performed in "young" and "old" larval lamprey that differed in age by at least one year. In old larval lamprey, significantly more descending brain neurons projected to specific rostral levels of the spinal cord than in young animals. Furthermore, in young and old lamprey, the main morphological change in Müller and Mauthner cells was an increase in soma size. The major conclusion from the present study is that in larval lamprey, some new brain-spinal cord projections are added with age that could be due to axonal elongation by preexisting brain neurons and/or descending projections from new neurons (i.e., neurogenesis or maturation of incompletely differentiated neurons). Following spinal cord transections, the numbers of descending projections were not significantly different than those in normal, unlesioned animals. Thus, some new descending projections are added with age, but at a relatively slow rate, and the rate does not appear to be affected significantly by spinal cord transections. Together, the present results and those from our recent double-labeling study suggest that following spinal cord transection in larval lamprey, axonal regeneration by descending brain neurons, rather than the relatively slow addition of new brain-spinal cord projections with age, probably accounts for the majority of restored projections and recovery of locomotor function

Increased NCAM-180 Immunoreactivity and Maintenance of L1 Immunoreactivity in Injured Optic Fibers of Adult Mice

The injury related expression of two axon-growth promoting cell adhesion molecules (CAMs), NCAM-180 which is developmentally downregulated and L1 which is regionally restricted, were compared in optic fibers in the adult mouse. The neuron-specific isoform of NCAM (NCAM-180) is present at very low levels in unlesioned adult optic axons. At 7 days after nerve crush, immunoreactivity was strongly and uniformly increased in optic axons within the nerve and throughout retina. Reactivity in surviving axons had returned to control levels at 4 weeks. To induce regrowth of adult retinal ganglion cell axons retinal explants were placed in culture. Strong NCAM-180 staining was observed on these regenerating optic axons. The neuronal cell adhesion molecule L1 is restricted to retina and to the unmyelinated segment of the optic nerve near the optic nerve head in unlesioned adult animals. Following nerve crush, L1 immunoreactivity was retained within retina and proximal nerve and novel staining was detected in the more distal segment of the optic nerve up to the lesion site where it persisted for at least eight months. The capacity of optic fibers to show increased NCAM-180 immunoreactivity and maintain L1 expression after a lesion may explain why these fibers exhibit relatively good potential for regeneration.

Increased Response to Intrastriatal L(+)-2-Amino-4-Phosphonobutyrate (L-AP4) in Unilateral 6-Hydroxydopamine-Lesioned Rats

Unilateral intrastriatal injection of the prototype Group III metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyrate (L-AP4) induces rotation in rats with ipsilateral unilateral 6-hydroxydopamine lesions of the nigrostriatal projection but not in unlesioned control animals [Kearney et al., (1998) Neuroscience 87:881–891]. We studied differences in striatal neuron Fos-like immunoreactivity expression, striatal neuron Fos-like immunoreactivity localization, and regional [14C]-2-deoxyglucose uptake after unilateral intrastriatal injection of L-AP4 between control and unilateral 6-hydroxydopamine lesioned rats. There was a left shift of the dose response curve with more striatal neurons expressing Fos-like immunoreactivity at lower doses of L-AP4 in 6-hydroxydopamine lesioned animals. In unlesioned striata, L-AP4 injections tended to induce Fos-like immunoreactivity in striato-pallidal projection neurons. In lesioned animals, the majority of striatal neurons expressing Fos-like immunoreactivity were striatonigral projection neurons. In both control and lesioned animals, intrastriatal injection of L-AP4 produced widespread decreases in [14C]-2-deoxyglucose uptake in basal ganglia nuclei and related regions, but the magnitude of this effect increased markedly in lesioned animals. Striatal dopamine denervation enhances the response of striatal neurons to intrastriatally injected L-AP4 with an apparent shift in the type of striatal projection neuron responding to L-AP4.

Compensatory Sprouting and Impulse Rerouting after Unilateral Pyramidal Tract Lesion in Neonatal Rats

After lesions of the developing mammalian CNS, structural plasticity and functional recovery are much more pronounced than in the mature CNS. We investigated the anatomical reorganization of the corticofugal projections rostral to a unilateral lesion of the corticospinal tract at the level of the medullary pyramid (pyramidotomy) and the contribution of this reorganization and other descending systems to functional recovery. Two-day-old (P2) and adult rats underwent a unilateral pyramidotomy. Three months later the corticofugal projections to the red nucleus and the pons were analyzed; a relatively large number of corticorubral and corticopontine fibers from the lesioned side had crossed the midline and established an additional contralateral innervation of the red nucleus and the pons. Such anatomical changes were not seen after adult lesions. Intracortical microstimulation of the primary motor cortex with EMG recordings of the elbow flexor muscles were used to investigate possible new functional connections from the motor cortex of the pyramidotomy side to the periphery. In rats lesioned as adults, stimulation of the motor cortex ipsilateral to the pyramidotomy never elicited EMG activity. In contrast, in P2 lesioned rats bilateral forelimb EMGs were found. EMG latencies were comparable for the ipsilateral and contralateral responses but were significantly longer than in unlesioned animals. Transient inactivation of both red nuclei with the GABA receptor agonist muscimol led to a complete loss of these bilateral movements. Movements and EMGs reappeared after wash-out of the drug. These results suggest an important role of the red nucleus in the reconnection of the cortex to the periphery after pyramidotomy.

The Expression of the Calcium Binding Protein Calretinin in the Rat Striatum: Effects of Dopamine Depletion and L-DOPA Treatment

The activity of the striatum is regulated by glutamate and dopamine neurotransmission. Consequent to striatal dopamine depletion the corticostriatal excitatory input is increased, which in turn can raise intracellular calcium levels. We investigated changes in the neuronal expression of the calcium binding protein calretinin related to dopamine depletion and l-DOPA administration. Immunohistochemical methods were used to assess calretinin in the striatum of rats with unilateral lesions of the nigrostriatal system. In these animals we observed a loss of the patchy distribution of calretinin fibers. Moreover, after dopaminergic depletion we detected two new, not previously described, calretinin cell types, the presence of which could be related to morphological changes induced by loss of a dopaminergic input. We also found an increase in the number of calretinin-labeled cells in the striatum ipsilateral to the lesion compared to the contralateral striatum or to the striatum of normal rats. This increase was mostly evident at 3 weeks postlesion and tended to decrease toward normal levels at 6, 10, and 18 weeks postlesion. In unlesioned animals, l-DOPA administration did not induce changes in the expression of calretinin. In unilaterally lesioned animals, l-DOPA reversed the increase in the number of calretinin-positive cells induced by the lesion. However, chronic l-DOPA administration was less effective than acute l-DOPA in reversing the effect of the lesion. The present data suggests that striatal calretinin neurons are sensitive to dopamine depletion. Increased expression of calretinin in striatal cells may be consequent to enhanced striatal excitatory input.

Outgrowth-promoting molecules in the adult hippocampus after perforant path lesion.

Lesion-induced neuronal plasticity in the adult central nervous system of higher vertebrates appears to be controlled by region- and layer-specific molecules. In this study we demonstrate that membrane-bound hippocampal outgrowth-promoting molecules, as present during the development of the entorhino-hippocampal system and absent or masked in the adult hippocampus, appear 10 days after transection of the perforant pathway. We used an outgrowth preference assay to analyse the outgrowth preference of axons from postnatal entorhinal explants on alternating membrane lanes obtained from hippocampus deafferented from its entorhinal input taken 4, 10, 20, 30 and 80 days post-lesion and from adult control hippocampus. Neurites from the entorhinal cortex preferred to extend axons on hippocampal membranes disconnected from their entorhinal input for 10 days in comparison with membranes obtained from unlesioned adult animals. Membranes obtained from hippocampi disconnected from their entorhinal input for 10 days were equally as attractive for growing entorhinal cortex (EC) axons as membranes from early postnatal hippocampi. Further analysis of membrane properties in an outgrowth length assay showed that entorhinal axons extended significantly longer on stripes of lesioned hippocampal membranes in comparison with unlesioned hippocampal membranes. This effect was most prominent 10 days after lesion, a time point at which axonal sprouting and reactive synaptogenesis are at their peak. Phospholipase treatment of membranes obtained from unlesioned hippocampi of adult animals strongly promoted the outgrowth length of entorhinal axons on these membranes but did not affect their outgrowth preference for deafferented hippocampal membranes. Our results indicate that membrane-bound outgrowth-promoting molecules are reactivated in the adult hippocampus following transection of the perforant pathway, and that neonatal entorhinal axons are able to respond to these molecules. These findings support the hypothesis of a temporal accessibility of membrane-bound factors governing the layer-specific sprouting of remaining axons following perforant path lesion in vivo.

Anatomic basis of sequence-dependent predictability exhibited by nigral dopamine neuron firing patterns.

Interspike intervals (ISIs) of dopamine (DA) neurons recorded in the substantia nigra are predicted partially by their immediate prior history. This study was designed to assess neuroanatomic origins of these sequential relationships. ISI data recorded from three groups of nigral DA neurons were studied: 1) 16 neurons recorded in unlesioned animals, 2) 14 neurons recorded after forebrain hemisection, 3) 12 neurons recorded after partial forebrain hemisection that reproduced nonspecific effects of the surgical lesion while leaving forebrain connections intact. As predicted, DA neurons recorded after full forebrain hemisection yielded statistically significant reductions in sequential predictability relative to control neurons and neurons recorded following partial hemisection. These data support the hypothesis that the sequence-dependent behavior of DA neurons arise in part from interactions with forebrain structures. ISI sequences recorded from unlesioned rats demonstrated maximum predictability when an average of 3.7 prior events were incorporated into the forecasting algorithm, thereby suggesting a physiological process whose "depth" of history-dependence is approximately 600-800 msec. Additional studies examining the functional significance of sequence-dependent ISI structure exhibited by nigral DA neurons are indicated.