Impaired glutamate homeostasis and programmed cell death in a chronic MPTP mouse model of Parkinson's disease.

The pathogenesis of Parkinson's disease is not fully understood, but there is evidence that excitotoxic mechanisms contribute to the pathology. However, data supporting a role for excitotoxicity in the pathophysiology of the disease are controversial and sparse. The goal of this study was to determine whether changes in glutamate signaling and uptake contribute to the demise of dopaminergic neurons in the substantia nigra. Mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid or vehicle (probenecid or saline alone). Extracellular levels of glutamate in the substantia nigra were substantially increased, and there was an increase in the affinity, but no change in the velocity, of glutamate transport after MPTP/probenecid treatment compared to vehicle controls. In addition, the substantia nigra showed two types of programmed death, apoptosis (type I) and autophagic (type II) cell death. These data suggest that increased glutamate signaling could be an important mechanism for the death of dopaminergic neurons and trigger the induction of programmed cell death in the chronic MPTP/probenecid model.

Cholinergic neurons of the adult rat striatum are immunoreactive for glutamatergic N-methyl-d-aspartate 2D but not N-methyl-d-aspartate 2C receptor subunits.

Cholinergic neurons of the striatum play a crucial role in controlling output from this region. Their firing is under the control of a relatively limited glutamatergic input, deriving principally from the thalamus. Glutamate transmission is effected via three major subtypes of receptors, including those with affinity for N-methyl-d-aspartate (NMDA) and the properties of individual receptors reflect their precise subunit composition. We examined the distribution of NMDA2C and NMDA2D subunits in the rat striatum using immunocytochemistry and show that a population of large neurons is strongly immunoreactive for NMDA2D subunits. From their morphology and ultrastructure, these neurons were presumed to be cholinergic and this was confirmed with double immunofluorescence. We also show that NMDA2C is present in a small number of septal and olfactory cortical neurons but absent from the striatum. Receptors that include NMDA2D subunits are relatively insensitive to magnesium ion block making neurons more likely to fire at more negative membrane potentials. Their localization to cholinergic neurons may enable very precise regulation of firing of these neurons by relatively small glutamatergic inputs.

Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1).

Three isoforms of a vesicular glutamate transporter (VGLUT1-3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down-regulation of VGLUT1 transporter on anxiety, depressive-like behaviour and learning. The behavioural phenotype of VGLUT1-heterozygous mice (C57BL/6) was compared to wild-type (WT) littermates. Moreover, VGLUT1-3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1-heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light-dark exploration test and depressive-like behaviour in the forced swimming test: no differences were shown in the elevated plus-maze model of anxiety. In the novel object recognition test, VGLUT1(+/-) mice showed normal short-term but impaired long-term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1-mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.

The anatomy of co-morbid neuropsychiatric disorders based on cortico-limbic synaptic interactions.

Many brain disorders appear to involve dysfunctions of aminergic systems. Alterations in dopamine activity may underpin both schizophrenia and the establishment and maintenance of drug dependence while disruption of serotonergic signalling may be crucial in both depression and schizophrenia. The co-existence of nicotine and alcohol abuse with depression and schizophrenia is well-documented as is the particular vulnerability of adolescents. At the same time, a common group of brain structures is increasingly implicated in neuropathological studies. For example, depression may involve a lack of serotonin signalling, particularly in the prefrontal cortex, while in schizophrenia there is evidence for reduced dopamine signalling in the same brain region, co-existing with hyperactivity in the mesolimbic dopamine pathway. Increased dopamine release from the mesolimbic dopamine pathway is also a common factor of drugs of abuse. Furthermore, the control of motivational behaviour and dopamine release is apparently modified by hippocampal and amygdala activity, both brain regions showing pathological changes in schizophrenia and depression. Our work has focused on the intricate synaptic interactions of aminergic terminals and cortical and subcortical neurons in order to unravel the anatomical basis for these disorders and their treatments. We show convergence of dopamine and cortical inputs onto single neurons in the nucleus accumbens, and between different cortical inputs to individual neurons, providing a basis for the gating mechanisms attributed to these interactions. We have also examined local and extrinsic connections in the prefrontal cortex and the basis for regulation of both cortical neurons and midbrain dopamine neurons by serotonin from the raph é nucleus. Together with data concerning subcellular receptor distributions, this information provides a detailed synaptic framework for interpreting behavioural, pharmacological and physiological data and enhances our understanding of possible circuitry underlying comorbidity of disorders such as schizophrenia and depression with drug abuse, information invaluable in the introduction of enhanced therapies.

Medial prefrontal cortex volume loss in rats with isolation rearing-induced deficits in prepulse inhibition of acoustic startle.

Rearing rats in isolation produces perturbations in behavior and brain neurochemistry suggested to resemble those of schizophrenia. In particular, isolation-reared rats display deficits in prepulse inhibition of acoustic startle that in humans are associated with disorders including schizophrenia and are interpreted as abnormalities in sensorimotor gating. The prefrontal cortex is considered important in the regulation of prepulse inhibition of acoustic startle and postmortem studies suggest that neuropil and total volume, but not total number of neurons, are decreased in this region of the brains of schizophrenic patients. In this study we used design-based stereological techniques to examine the brains of Lister Hooded rats, reared in isolation and which displayed prepulse inhibition of acoustic startle deficits, for changes in morphology compared with the brains of their socially-reared littermates. Pooled data from three batches of animals revealed a significant 7% volume loss of the medial prefrontal cortex of isolation-reared rats whereas neuron number in this region was unchanged. In contrast, volume and total neuron number were unaffected in the rostral caudate putamen. The robust reduction in prefrontal cortical volume observed in isolation-reared rats, in the absence of reductions in total neuron number, suggest that there is a loss of volume of the neuropil. These changes parallel those reported in schizophrenia patients and therefore support the construct validity of this model.

Nucleus accumbens nitric oxide immunoreactive interneurons receive nitric oxide and ventral subicular afferents in rats.

The nitric oxide generating neurons of the nucleus accumbens exert a powerful influence over striatal function, in addition, these nitrergic inputs are in a position to regulate the dopaminergic and glutamatergic inputs on striatal projection neurons. It was the aim of this study to establish the source of the glutamatergic drive to nitric oxide synthase interneurons of the nucleus accumbens. The nucleus accumbens nitric oxide-generating neurons receive asymmetrical, excitatory, presumably glutamatergic inputs. Possible sources of these inputs could be the limbic and cortical regions known to project to this area. To identify sources of the excitatory inputs to the nitric oxide synthase-containing interneurons of the nucleus accumbens in the rat we first examined the ultrastructural morphology of asymmetrical synaptic specializations contacting nitric oxide synthase-immunohistochemically labeled interneurons in the nucleus accumbens. Neurons were selected from different regions of the nucleus accumbens, drawn using camera lucida, processed for electron microscopic analysis, and the boutons contacting nitric oxide synthase-labeled dendrites were photographed and correlated to the drawings. Using vesicle size as the criterion the source was predicted to be either the prefrontal cortex or the ventral subiculum of the hippocampus. To examine this prediction, a further study used anterograde tracing from both the prefrontal cortex and the ventral subiculum, and nitric oxide synthase immunohistochemistry with correlated light and electron microscopy. Based on appositions by anterogradely labeled fibers, selected nitric oxide synthase-labeled neurons within the nucleus accumbens, were examined with electron microscopic analysis. With this technique we confirmed the prediction that subicular afferent boutons make synaptic contact with nitric oxide synthase interneurons, and demonstrated anatomically that nitric oxide synthase boutons make synaptic contact with the dendritic arbors of nitric oxide synthase interneurons. We suggest that the subicular input may excite the nitric oxide synthase neurons synaptically, while the nitric oxide synthase-nitric oxide synthase interactions underlie a nitric oxide signaling network which propagates hippocampal information, and expands the hippocampus's influence on 'gating' information flow across the nucleus accumbens.

Localization of alpha-synuclein to identified fibers and synapses in the normal mouse brain.

Alpha-synuclein is a synaptic associated protein that is found throughout the brain. Although its function is not fully understood, various roles have been proposed, including the mobilization of synaptic vesicles and plasticity. However, interest in this molecule is mainly focused on its role in neurodegenerative diseases such as Parkinson's disease, where it is a major component in cellular inclusions. Although it is widely accepted that alpha-synuclein is distributed to terminals and fibers throughout the brain, the identity of the pathways that contain this protein is not known. To address this issue, we combined immunocytochemistry with anterograde tract-tracing in mouse to identify the projections that are alpha-synuclein immunopositive. We find that it is present in corticostriatal, nigrostriatal and striatonigral terminals. Our data support the concept that alpha-synuclein is normally present in at least some of the terminals of inclusion-forming neurons, but that it is also present in the axonal boutons of neurons that do not apparently accumulate this protein pathologically.

Individual nucleus accumbens-projection neurons receive both basolateral amygdala and ventral subicular afferents in rats.

The nucleus accumbens is regarded as the limbic-motor interface, in view of its limbic afferent and somatomotor and autonomic efferent connections. Within the accumbens, there appear to be specific areas in which limbic afferent fibres, derived from the hippocampus and the amygdala, overlap. These afferent inputs have been suggested to converge monosynaptically on cells within the accumbens and are hypothesized to play a role in paradigms such as conditioned place preference. Convergence between inputs from basolateral amygdala and hippocampus can be demonstrated with electrophysiological recording methods, but these do not conclusively preclude polysynaptic mechanisms. We examined the synaptic input to the projection neurons of the accumbens, the medium-sized densely spiny neurons. We labelled the projection neurons with a small injection of biotinylated dextran amine into the accumbens, and the afferents from the basolateral amygdala and ventral subiculum of the hippocampus with injections of biotinylated dextran amine and Phaseolus vulgaris-leucoagglutinin respectively, and revealed the anterogradely labelled fibres with different chromogens. The labelled accumbens-projection neurons were studied with correlated light and electron microscopy for identified monosynaptic inputs. With this technique we have demonstrated anatomically that monosynaptic convergence between the ventral subicular region of the hippocampus and the basolateral region of the amygdala occurs at the level of the proximal as well as distal dendrites. Finally, we suggest that these anatomical arrangements may represent the framework for the integrative role that has been assigned to the accumbens.

Lysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson's disease.

We have detected granular and filamentous inclusions that are alpha-synuclein- and ubiquitin-immunoreactive in the cytoplasm of dopaminergic and cortical neurons of C57/black mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid. The immunoreactive aggregates only become evident several weeks after large-scale dopaminergic cell death and a downregulation of alpha-synuclein gene expression. Numerous lipofuscin granules accumulate alpha-synuclein in the nigral and limbic cortical neurons of treated mice. These data provide evidence that insoluble proteins, such as alpha-synuclein, build up as granular and filamentous inclusions in dopaminergic neurons that survive the initial toxic MPTP insult. They further suggest that defective protein degradation rather than altered gene expression underlies deposition of alpha-synuclein and that abundant lysosomal compartments are present to seal off the potentially toxic material.

Mouse model of Parkinsonism: a comparison between subacute MPTP and chronic MPTP/probenecid treatment.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used to induce an animal model of Parkinsonism. The conventional mouse model, which usually involves acute or subacute injections of MPTP, results in a significant but reversible loss of dopaminergic functions. We have developed an alternative mouse model, in which co-administration of MPTP with probenecid results in the chronic loss of striatal dopamine for at least 6 months after cessation of treatment. In the present study, we compare the neurochemical, morphological and behavioral changes that occur in this alternative, chronic model with those in the conventional, subacute model. In the chronic model, we demonstrate an almost 80% loss of striatal dopamine and dopamine uptake 6 months after withdrawal from treatment. The neurochemical signs match unbiased stereological measures that demonstrate gradual loss of substantia nigra neurons. Rotarod performance further substantiates these findings by showing a progressive decline in motor performance. Based on the comparisons made in this study in mice, the chronic MPTP/probenecid model shows considerable improvements over the conventional, subacute MPTP model. The sustained alterations in the nigrostriatal pathway resemble the cardinal signs of human Parkinson's disease and suggest that this chronic mouse model is potentially useful to study the pathophysiology and mechanisms of Parkinsonism. It should also prove useful for the development of neuroprotection strategies.

Differences in the laminar origin of projections from the medial prefrontal cortex to the nucleus accumbens shell and core regions in the rat.

The medial prefrontal cortex (mPFC) projects to the nucleus accumbens shell, core and rostral pole. In this retrograde tract-tracing study of rat mPFC to nucleus accumbens projection neurons, the advantages of Neurobiotin are utilised in order to reveal the detailed morphology of labelled projection cells, and to permit an examination of the laminar projections to shell and core compartments The retrogradely transported Neurobiotin was found in somata, proximal and distal dendrites of neurons that project from the mPFC to the nucleus accumbens. The morphology of these projection neurons was revealed in great detail and confirmed that the projection arises wholly from pyramidal cells. Interestingly, it was also found that retrogradely labelled neurons were exclusively located in prelimbic and infralimbic regions in layers V and VI, after shell injections, but also in layer II following core sites. This observation may reflect possibly different roles for cortical laminae on the nucleus accumbens.

Structural and functional abnormalities of the hippocampal formation in rats with environmentally induced reductions in prepulse inhibition of acoustic startle.

The effects of social isolation on prepulse inhibition of acoustic startle (PPI), electrophysiology and morphology of subicular pyramidal neurons and the densities of interneuronal sub-types in the hippocampal formation were examined. Wistar rats (male weanlings) were housed socially (socials, n=8) or individually (isolates, n=7). When tested eight weeks later, PPI was lower in isolates. Rats then received terminal anaesthesia before slices of hippocampal formation were made in which the electrophysiological properties of a total of 108 subicular neurons were characterized. There were no differences in neuronal sub-types recorded in socials compared with isolates. Intrinsically burst-firing and regular spiking pyramidal neurons were examined in detail. There were no differences in resting membrane potential or input resistance in isolates compared with socials but action potential height was reduced and action potential threshold raised in isolates. A limited morphological examination of Neurobiotin-filled intrinsically burst-firing neurons did not reveal differences in cell-body area or in number of primary dendrites. Sections from the contralateral hemispheres of the same rats were stained with antibodies to calretinin, parvalbumin and the neuronal isoform of nitric oxide synthase (nNOS). In isolates, the density of calretinin positive neurons was increased in the dentate gyrus but unchanged in areas CA3, CA1 and subiculum. Parvalbumin and nNOS positive neuronal densities were unchanged. Hence in rats with environmentally induced reductions in PPI there are structural and functional abnormalities in the hippocampal formation. If the reduction in PPI stems from these abnormalities, and reduced PPI in rats is relevant to schizophrenia, then drugs that correct the reported electrophysiological changes might have antipsychotic effects.

Ultrastructural features of the nitric oxide synthase-containing interneurons in the nucleus accumbens and their relationship with tyrosine hydroxylase-containing terminals.

The ultrastructural features of neuronal nitric oxide synthase (NOS) -immunoreactive interneurons of rat nucleus accumbens shell and core were studied and compared. The NOS-containing subpopulation displayed characteristics similar to those previously described for nicotinamide adenine dinucleotide phosphate diaphorase-, neuropeptide Y, or somatostatin-containing striatal neurons, but also showed properties not previously associated with them, particularly the formation of both asymmetric and symmetric synaptic junctions. Inputs derived mainly from unlabeled terminals, but some contacts were made by NOS-immunolabeled terminals, by means of asymmetric synapses. Immunopositive endings that formed symmetric synapses were mainly onto dendritic shafts, whereas those that formed asymmetric synapses targeted spine heads. Morphometric analysis revealed that the core and shell NOS-stained neurons had subtly different innervation patterns and that immunostained terminals were significantly larger in the shell. A parallel investigation explored synaptic associations with dopaminergic innervation identified by labeling with an antibody against tyrosine hydroxylase (TH). In both shell and core, TH-positive boutons formed symmetric synapses onto NOS-containing dendrites, and in the core, TH- and NOS-immunolabeled terminals converged on both a single spiny dendrite and a spine. These results suggest that, in the rat nucleus accumbens, NOS-containing neurons may be further partitioned into subtypes, with differing connectivities in shell and core regions. These NOS-containing neurons may be influenced by a dopaminergic input. Recent studies suggest that nitric oxide potentiates dopamine release and the current study identifies the medium-sized, densely spiny neurons as a possible site of such an interaction.

Immunocytochemical characterization of catecholaminergic neurons in the rat striatum following dopamine-depleting lesions.

It is possible either permanently or transiently to deplete the rat striatum of dopamine. Following such depletions, striatal neurons immunoreactive for tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC) or dopamine appear. The presence of dopamine-producing neurons in the striatum has relevance for the treatment of Parkinson's disease, but whether these catecholaminergic phenotypes all produce dopamine is unclear. In the present study we establish that after unilateral 6-hydroxydopamine lesions or methamphetamine administration, striatal TH-immunoreactive neurons differ in size, morphology and location from those that are immunopositive for AADC or dopamine. The TH-positive cells which were localized either to ventral parts of the striatum or to the central and dorsal areas of the caudate-putamen generally have the morphological features of projection neurons, whereas those containing AADC or dopamine were confined to subcallosal positions in the dorsal medial quadrant of the caudate-putamen and resemble small, local-circuit neurons. The fact that AADC-immunoreactive neurons overlap in size, morphology and location with the cells that produce dopamine suggests strongly that this population is dopaminergic. However, the simultaneous appearance of neurons that contain the TH enzyme but clearly do not make dopamine raises questions about the functional role of these cells and the cellular mechanisms responsible for their induction following striatal dopamine loss.

Comparisons of the densities of NADPHd reactive and nNOS immunopositive neurons in the hippocampus of three age groups of young nonhandled and handled rats.

The complete absence of handling of male rats during neonatal development (from birth to postnatal day 21) correlates with an impairment of latent inhibition [J. Feldon, I. Weiner, From an animal model of an attentional deficit towards new insights into the pathophysiology of schizophrenia, J. Psychiatr. Res. 26 (1992) 345-366.]. Such nonhandling of rats reportedly also correlates with a decreased expression of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) reactivity in the hippocampus in adult rats (6 months of age) when compared with rats of the same age that were handled during the same neonatal period [R.R. Vaid, B.K. Yee, U. Shalev, J.N. Rawlins, I. Weiner, J. Feldon, S. Totterdell, Neonatal nonhandling and in utero prenatal stress reduce the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon's horn of rats, J. Neurosci. 17 (1997) 5599-5609.]. The present study investigated whether such a decrease in NADPHd activity would be detectable at earlier ages. Therefore, the present study assessed the density of nitric oxide (NO) producing neurons in the fascia dentata and Ammon's horn in 28-, 54-, and 118-day-old nonhandled and handled male rats using NADPHd histochemistry and immunohistochemical localization of neuronal isoform of nitric oxide synthase (nNOS), a NADPHd. This showed that in these three age groups, the numbers of NADPHd positive neurons per unit area throughout the hippocampus of rats that received no handling during neonatal development did not differ significantly from those of rats that received regular daily handling. In addition, we found in the rats of 118 days of age that the areal density of nNOS immunopositive neurons in the hippocampus also did not differ significantly between nonhandled and handled rats. Nevertheless, in a parallel study, rats from the same experimental group receiving identical treatments showed the expected impairment of latent inhibition at 4 months of age [R. Weizman, J. Lehmann, S. Leschiner, I. Allmann, T. Stoehr, C. Heidbreder, A. Domeney, J. Feldon, M. Gavish, Long-lasting effect of early handling on the peripheral-type benzodiazepine receptor, Pharmacol. Biochem. Behav. in press.]. These results suggest that nonhandling of rats during the early neonatal period, that does result in impairment in latent inhibition, does not affect the numbers of NO producing neurons in the hippocampus in rats of young ages, including the age of observed impairment of latent inhibition.

A comparison of the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon's horn between 6-month and 12-month old dark agouti rats.

The present study aimed to assess the developmental progress of the hippocampal nitric oxide (NO) system within adulthood by comparing the density of NO-producing neurons in the fascia dentata and Ammon's horn in two groups of adult male rats using NADPH-diaphorase (NADPH-d) histochemistry. One group comprised 6-month-old rats (early adulthood), and the other 12-month-old rats (middle-adulthood). Areal density (number of neurons per unit area) of NADPH-d positive neurons along the three hippocampal axes (septo-temporal, transverse and radial axes) were subjected to quantitative analyses. There were significant variations in the density of NADPH-d-reactive neurons along the transverse and radial axes of the hippocampus, similar to what have been described previously. Comparison between 6-month and 12-month-old rats indicated a substantial reduction in the density of NADPH-d-reactive neurons in the fascia dentata (69%) and Ammon's horn (54%) of the latter group. This reduction was relatively uniform along the septotemporal and radial axes, but appeared to be more pronounced in the fascia dentata and in the proximal region of Ammon's horn. Our finding showed that the hippocampal NO system can undergo significant changes within adulthood. It further highlighted the possibility that an age-related reduction in the capacity to produce NO may not be directly responsible for the cognitive decline associated with senescence, but rather predisposes neuronal degeneration in later life.

Light and electron microscopic study of neuronal nitric oxide synthase-immunoreactive neurons in the rat subiculum.

Neurons in the rat subiculum that are capable of producing nitric oxide were studied by using an antibody to the neuronal isoform of nitric oxide synthase (nNOS). In the light microscope, the staining pattern with the nNOS antibody closely resembled that seen following histochemical processing with nicotinamide adenine dinucleotide phosphate diaphorase. Immunostained neurons were found in all layers, and, in addition, large dendrites in the apical dendrite layer were also immunopositive. Although a few immunolabelled cells had the typical morphology of interneurons, most were found to have the characteristics of pyramidal neurons. In the subiculum, these immunoreactive pyramidal neurons were concentrated mainly in the most superficial cell layers and closest to the CA1 region, but pyramidal neurons in the CA1 layer of the hippocampus were consistently immunonegative. Immunopositive profiles in the subiculum were studied in the electron microscope and compared with unlabelled structures. Ultrastructural criteria suggest that both pyramidal and nonpyramidal subicular neurons are immunopositive for nNOS. Large, spiny dendrites and smaller, varicose dendrites were found to be immunoreactive for nNOS. Vesicle-containing profiles were probably presynaptic axons, and immunopositive boutons were seen to make symmetrical and asymmetrical synaptic contacts.

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro.

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.

Neonatal nonhandling and in utero prenatal stress reduce the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon's horn of rats.

The density of nitric oxide (NO)-producing neurons in the fascia dentata and Ammon's horn was assessed in 6-month-old male rats using NADPH-diaphorase (NADPH-d) histochemistry. Two separate experiments investigated whether (1) the complete absence of neonatal handling or (2) the administration of periodic prenatal stress could affect the expression and distribution of NADPH-d reactivity in the hippocampus, when compared with rats raised in normal standard laboratory conditions. Experiment 1 demonstrated that adult rats that received no handling during neonatal development (from birth to postnatal day 22) showed a very substantial reduction in NADPH-d-positive neurons per unit area throughout the entire hippocampus when compared with rats that received regular daily handling in this period. Quantitative analysis further revealed that this effect was significantly more pronounced in Ammon's horn than in the fascia dentata, and within Ammon's horn the dorsal region was selectively more affected. Experiment 2 showed that prenatal stress, which involved the administration of daily restraint stress to pregnant dams throughout the gestation period, also led to a reduction in NADPH-d reactivity in the hippocampus of the offspring of these dam when they reached adulthood. The present results suggest that behavioral manipulations in the early neonatal or prenatal period can significantly alter the neurodevelopment of the hippocampal NO system and these changes might be related to some of the behavioral abnormalities that emerge later in adulthood.

Topographical organization of projections from the entorhinal cortex to the striatum of the rat.

The efferent projections of the entorhinal cortex to the striatum were studied with retrograde (horseradish peroxidase wheat germ agglutinin) and anterograde (biocytin and biotinylated dextran amine) tracing methods. The bulk of the entorhinal cortical fibres were found to project to the nucleus accumbens in the ventral striatum, but the caudate putamen is only sparsely and diffusely innervated, rostrally, along its dorsal and medial borders. Fibres arising from neurons in the lateral entorhinal cortex project throughout the rostrocaudal extent of the nucleus accumbens but are most abundant in the core and lateral shell of that nucleus. The rostral neurons of the medial entorhinal cortex were found to project sparsely to the striatum, whereas caudal neurons provide a dense input to the rostral one-third of the nucleus accumbens, especially to the rostral pole, where they concentrate more in the core than in the shell. Contralateral entorhinal projections, which are very sparse, were found in the same parts of the nucleus accumbens and the caudate-putamen as the ipsilateral terminal fields. The present observations that entorhinal inputs to the nucleus accumbens are regionally aligned suggest that disruption of these connections could produce site-specific deficits with, presumably, specific behavioural consequences.