Post synaptic density protein 95 (PSD-95) is a postsynaptic adaptor protein coupling the NMDA receptor to downstream signalling pathways underlying plasticity. Mice carrying a targeted gene mutation of PSD-95 show altered behavioural plasticity including spatial learning, neuropathic pain, orientation preference in visual cortical cells, and cocaine sensitisation. These behavioural effects are accompanied by changes in long-term potentiation of synaptic transmission. In vitro studies of PSD-95 signalling indicate that it may play a role in regulating dendritic spine structure. Here, we show that PSD-95 mutant mice have alterations in dendritic spine density in the striatum (a 15% decrease along the dendritic length) and in the hippocampus (a localised 40% increase) without changes in dendritic branch patterns or gross neuronal architecture. These changes in spine density were accompanied by altered expression of proteins known to interact with PSD-95, including NR2B and SAP102, suggesting that PSD-95 plays a role in regulating the expression and activation of proteins found within the NMDA receptor complex. Thus, PSD-95 is an important regulator of neuronal structure as well as plasticity in vivo.

Dendritic spines are important structures which receive synaptic inputs in many regions of the CNS. The goal of this study was to test the hypothesis that numbers of dendritic spines are significantly reduced on spiny neurones in basal ganglia regions in Parkinson's disease as we had shown them to be in a rat model of the disease [Exp Brain Res 93 (1993) 17]. Postmortem tissue from the caudate and putamen of patients suffering from Parkinson's disease was compared with that from people of a similar age who had no neurological damage. The morphology of Golgi-impregnated projection neurones (medium-sized spiny neurones) was examined quantitatively. The numerical density of dendritic spines on dendrites was reduced by about 27% in both nuclei. The size of the dendritic trees of these neurones was also significantly reduced in the caudate nucleus from the brains of PD cases and their complexity was changed in both the caudate nucleus and the putamen. Dendritic spines receive crucial excitatory input from the cerebral cortex. Reduction in both the density of spines and the total length of the remaining dendrites is likely to have a grave impact on the ability of these neurones to function normally and may partly explain the symptoms of the disorder.

We carried out an ultrastructural analysis of axotomized synaptic terminals in Wld s and Ube4b/Nmnat ( Wld ) transgenic mice, in which severed distal axons are protected from Wallerian degeneration. Previous studies have suggested that axotomy in juvenile (< 2 months) Wld mice induced a progressive nerve terminal withdrawal from motor endplates. In this study we confirm that axotomy‐induced terminal withdrawal occurs in the absence of all major ultrastructural characteristics of Wallerian degeneration. Pre‐ and post‐synaptic membranes showed no signs of disruption or fragmentation, synaptic vesicle densities remained at pre‐axotomy levels, the numbers of synaptic vesicles clustered towards presynaptic active zones did not diminish, and mitochondria retained their membranes and cristae. However, motor nerve terminal ultrastructure was measurably different following axotomy in Wld transgenic 4836 line mice, which strongly express Wld protein: axotomized presynaptic terminals were retained, but many were significantly depleted of synaptic vesicles. These findings suggest that the Wld gene interacts with the mechanisms regulating transmitter release and vesicle recycling.

It has been shown that dendritic spine density of medium sized spiny neurons in the neostriatum is reduced by about 19% in an animal model of Parkinson's disease (unilateral 6-hydroxydopamine lesion in rats) (Ingham el al., 1993). Recent results show an even greater reduction of spines (>20%) in the caudate and putamen on these neurons in Parkinson's disease patients. The spines receive input from axons originating mainly from the cortex, which form asymmetric synapses. Stimulation of these corticostriatal fibres leads to excitation of neostriatal neurons in a manner modifiable by dopamine. A similar reduction of asymmetric synapses (19%) in the neostriatum deprived of dopaminergic input compared with the intact side (Ingham et al., 1998) suggests this excitatory input is lost along with the spines. In the same study it was found that the numerical density of a sub-population of asymmetric synapses with discontinuous or complex synaptic specialisations was significantly increased. Such synapses would be expected to have increased efficacy and may be the morphological correlate of the proposed increase in excitation into the neostriatum in Parkinson's disease. A second unbiased, stereological study of the animal model suggests that both changes occur on neurons projecting indirectly via the globus pallidus to output nuclei of the basal ganglia.

After the unilateral destruction of the dopamine input to the neostriatum there are enduring changes in rat behaviour. These have been ascribed to the loss of dopamine and the animals are often referred to as ‘hemiparkinsonian’. In the denervated neostriatum, we have shown that not only are the tyrosine hydroxylase positive boutons missing, but also the medium sized densely spiny output cells have fewer spines. Spines usually have asymmetric synapses on their heads. In a recent stereological study we were able to show that there is a loss of approximately 20% of asymmetric synapses in the lesioned neostriatum by 1 mo after the lesion. Current experiments are trying to establish the specificity of this loss. So far we have evidence suggesting that there is no obvious preferential loss of synapses from either D1 or D2 receptor immunostained dendrites in the neostriatum with damaged dopamine innervation. These experiments suggest that dopamine is somehow necessary for the maintenance of corticostriatal synapses in the neostriatum. In a different series of experiments slices of cortex and neostriatum were maintained in vitro in such a way as to preserve at least some of the corticostriatal connections. In this preparation we have been able to show that cortical stimulation results in robust excitatory postsynaptic potentials (EPSPs) recorded from inside striatal neurons. Using stimulation protocols derived from the experiments on hippocampal synaptic plasticity we have shown that the usual consequence of trains of high frequency stimulation of the cortex is the depression of the size of EPSPs in the striatal cell. In agreement with similar experiments by others, the effect seems to be influenced by NMDA receptors since the unblocking of these receptors with low Mg++ concentrations in the perfusate uncovers a potentiation of the EPSPs after trains of stimulation. Dopamine applied in the perfusion fluid round the slices has no effect but pulsatile application of dopamine, close to the striatal cell being recorded from, and in temporal association with the cortical trains, leads to a similar LTP like effect. The reduction of K+ channel conductance in the bath with TEA also has the effect of making cortical trains induce potentiation of corticostriatal transmission. TEA applied only to the cell being recorded from has no similar effect; the cortical stimulation again depresses the EPSP amplitude, so the site of action of TEA may well be presynaptic to the striatal cell. The morphological and physiological experiments may not necessarily be related but it is tempting to suggest that dopamine protects some corticostriatal synapses by potentiating them but that in the absence of dopamine others simply disconnect and are no longer detectable on electron microscopy.

After the unilateral destruction of the dopamine input to the neostriatum there are enduring changes in rat behaviour. These have been ascribed to the loss of dopamine and the animals are often referred to as 'hemiparkinsonian'. In the denervated neostriatum, we have shown that not only are the tyrosine hydroxylase positive boutons missing, but also the medium sized densely spiny output cells have fewer spines. Spines usually have asymmetric synapses on their heads. In a recent stereological study we were able to show that there is a loss of approximately 20% of asymmetric synapses in the lesioned neostriatum by 1 mo after the lesion. Current experiments are trying to establish the specificity of this loss. So far we have evidence suggesting that there is no obvious preferential loss of synapses from either D1 or D2 receptor immunostained dendrites in the neostriatum with damaged dopamine innervation. These experiments suggest that dopamine is somehow necessary for the maintenance of corticostriatal synapses in the neostriatum. In a different series of experiments slices of cortex and neostriatum were maintained in vitro in such a way as to preserve at least some of the corticostriatal connections. In this preparation we have been able to show that cortical stimulation results in robust excitatory postsynaptic potentials (EPSPs) recorded from inside striatal neurons. Using stimulation protocols derived from the experiments on hippocampal synaptic plasticity we have shown that the usual consequence of trains of high frequency stimulation of the cortex is the depression of the size of EPSPs in the striatal cell. In agreement with similar experiments by others, the effect seems to be influenced by NMDA receptors since the unblocking of these receptors with low Mg++ concentrations in the perfusate uncovers a potentiation of the EPSPs after trains of stimulation. Dopamine applied in the perfusion fluid round the slices has no effect but pulsatile application of dopamine, close to the striatal cell being recorded from, and in temporal association with the cortical trains, leads to a similar LTP like effect. The reduction of K+ channel conductance in the bath with TEA also has the effect of making cortical trains induce potentiation of corticostriatal transmission. TEA applied only to the cell being recorded from has no similar effect; the cortical stimulation again depresses the EPSP amplitude, so the site of action of TEA may well be presynaptic to the striatal cell. The morphological and physiological experiments may not necessarily be related but it is tempting to suggest that dopamine protects some corticostriatal synapses by potentiating them but that in the absence of dopamine others simply disconnect and are no longer detectable on electron microscopy.

The sensory input to the neostriatum from groups of cortical cells related to individual facial vibrissae has been investigated at both light- and electron-microscopic resolution. The purpose of the study was to establish the extent to which corticostriatal input maintains the anatomical coding of spatial information that is present in cortex. A double anterograde tracing method was used to identify the output projections from groups of adjacent neurons in different barrel columns, so that the anatomical relationships between two groups could be studied throughout their length. Adjacent whiskers are represented in adjoining cortical barrels and an examination of corticostriatal projections from these reveals two patterns of projection. In one, the anatomical topography is partially preserved; the barrels are represented in adjoining, discrete, areas of the somatosensory neostriatum. In the second projection pattern, the neostriatal innervation is diffuse and adjacent barrels are represented in overlapping regions of the neostriatum. Moreover, the fibres are thinner, have smaller boutons, and are present in both the ipsilateral and contralateral neostriatum. The two systems also enter the neostriatal neuropile separately. The discrete topographic system enters the adjacent neostriatum as collaterals which leave the descending corticofugal fibres at right angles, while the diffuse system enters directly from the corpus callosum at an acute angle. Examination of the neostriatal terminal fields by correlated light and electron microscopy, shows that characteristic axospinous terminals on spiny neurons are made by both groups of cortical fibres, although they differ in their size and morphology. It is concluded that at least two corticostriatal pathways arise from the barrel cortex. One connection maintains some of the anatomical code implicit in the barrel pattern of primary somatosensory cortex, but another, more diffuse, system is overlaid upon it which may carry different information from this complex area of cortex.

In the 6-hydroxydopamine model of Parkinson's disease in the rat, there is a significant reduction in the number of dendritic spines on the principal projection neurons in the neostriatum, presumably attributable to loss of the nigrostriatal dopamine input. These spines invariably receive input from terminals forming asymmetric synapses that originate mainly from the cortex. The object of the present study was to determine the fate of those terminals after the loss of dendritic spines. Unbiased estimates of synaptic density and absolute numbers of synapses in a defined volume of the neostriatum were made using the "disector" and Cavalieri techniques. Numerical synaptic density of asymmetric synaptic contacts was 17% lower in the neostriatum deprived of dopamine innervation and, in absolute terms, there were 3 billion (19%) fewer contacts. The numerical density of a subpopulation of asymmetric contacts on dendritic spines that have complex or perforated synaptic specializations and normally make up 9% of the asymmetric population was 44% higher on the experimental side. Asymmetric synapses were found to be enriched in glutamate using postembedding immunogold labeling. The present observations demonstrate that the loss of spines previously reported after 6-hydroxydopamine lesions is accompanied by a loss of asymmetric synapses rather than by the movement of synapses from spines to other postsynaptic targets. The study also demonstrates that there is an increase in complex synaptic interactions that have been implicated in synaptic plasticity in other regions of the CNS after experimental manipulations.

Ultrastructural studies have shown that dopamine synapses are localized to the necks of dendritic spines on striatal neurons. The researchers have examined the rat neostriatum in the electron microscope to investigate whether the increase in enkephalin that follows dopamine destruction, resulted from sprouting of enkephalin-containing terminal onto the spine neck sites vacated by dopamine. Enkephalin-immunoreactive synaptic boutons have been 50% larger after dopamine denervation, but they did not contact spines more frequently than in the control striatum. Detailed spine counting revealed that the number of dendritic spines on the output cells of the striatum, from which the dopamine has been removed by a 6-hydroxydopamine injection, is reduced. In a recent study using "unbiased" stereological methods to count the number of asymmetric synapses the total number of asymmetric synapses in the striatum on the lesioned side has been reduced by about 18% that is similar to the reduction in spine density at equivalent times after the lesion. The synapses that disappear after dopamine depletion might be the most vulnerable of a system of corticostriatal synapses maintained somehow by dopamine. Thus, taking the morphological and physiological experiments together suggests that long term potentiation (LTP) induced by dopamine has morphological consequences. It could be potentiated synapses survive and in the absence of dopamine, nonpotentiated synapses are eliminated. Testing these ideas will require more detail about how potentiation happens and some pharmacological tools with which to interfere with various stages of it.

In Parkinson’s disease the dopaminergic nigrostriatal pathway degenerates, resulting in an imbalance in activity of two pathways of information flow through the basal ganglia. In animal models of the disease, the striatonigral pathway becomes underactive and the striatopallidal pathway becomes overactive. In the present study immunocytochemistry for enkephalin and GABA and anterograde labelling were used to investigate whether morphological plasticity occurs in striatopallidal terminals following unilateral removal of the nigrostriatal dopaminergic pathway. Pallidal terminals were immunostained to reveal enkephalin and examined in the electron microscope ( n =399). Immunoreactive synaptic bouton profiles were on average 64% larger on the experimental side 26 days after the lesion. Analysis of their shape revealed that those on the dopamine-depleted side of the brain were more irregular in profile and that their synaptic specialisations were more complex in shape but not significantly different in length. Striatopallidal terminals were also identified by GABA immunocytochemistry combined with anterograde labelling ( n =20). Double-labelled boutons were significantly larger in cross-sectional area on the experimental side (57%). Analysis of terminals that were simply labelled by the immunogold method to reveal GABA ( n =278) showed no significant differences in size between terminals from the dopamine-depleted and control side. This suggests that a substantial number of GABAergic terminals in the globus pallidus do not belong to the striatopallidal population of terminals. These morphological changes correlate with previous studies suggesting striatopallidal boutons are more active after destruction of dopaminergic input to the neostriatum.

The morphological plasticity of an identified population of synaptic boutons in the rat neostriatum was investigated 24 h (short-term treatment) or 14 days (long-term treatment) after administration of the depot neuroleptic, haloperidol decanoate. Specific methionine(5)-enkephalin antiserum was used to label bouton profiles in the dorsal neostriatum. The size and shape of these boutons was subsequently analysed with quantitative methods at the ultrastructural level. Immunoreactive synaptic bouton profiles were found to have a larger cross-sectional area, to be less circular in shape and to have a longer maximum diameter after long-term neuroleptic treatment. These parameters were not significantly affected by short-term neuroleptic treatment. The morphological parameters indicate that methionine(5)-enkephalin-immunoreactive boutons become enlarged, probably by elongating. This suggests that boutons containing methionine(5)-enkephalin increase their potential synaptic efficacy in the long term after neuroleptic treatment.

Synapses of optic afferents (optic synapses) in the suprachiasmatic nucleus of hooded rats were morphometrically evaluated after exposing the animals to 12 h, 14 days, 2 months, and 8 months of constant light (light rats) and darkness (dark rats). Compared with dark rats, optic synapses from light rats have larger boutons with larger mitochondria, more clear vesicles, fewer dense-core vesicles and front-line vesicles, smaller presynaptic dense projections, a smaller amount of postsynaptic density material, a smaller relative number of Gray-type I (asymmetric) junctions, a greater relative number of Gray-type II (symmetric) junctions, as well as more and larger mitochondria in the postsynaptic dendrites. Junctions of optic synapses are mostly straight, but the small number of positively curved contacts are more flattened in light rats than in dark rats. An age-related increase in the size of presynaptic dense projections was also observed. There are no changes in the sizes of clear and dense-core vesicles, in the size of synaptic junctions and their numerical density in area, and in the unspecific contact area between pre- and postsynaptic elements. The changes in optic boutons are characteristic for activated and relatively disused synapses with a slow, tonic firing rate. It appears that (1) the amount of postsynaptic density material is proportional to the strength of Gray-type I synapses, and that (2) some excitatory optic synapses become inhibitory after long-term activity, whereas some inhibitory synapses turn into excitatory contacts after long-term disuse.

Parkinson's disease is characterised by the loss of dopaminergic neurons in the substantia nigra, pars compacta which normally provide dopaminergic input to the caudate nucleus and putamen (neostriatum). One of the well used 'animal models' of the disorder involves unilateral destruction of the dopaminergic pathway by injecting the toxin, 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle where it is taken up selectively by dopaminergic axons resulting in the death of all parts of the neuron (Ungerstedt and Arbuthnott, 1970). Several morphological changes have been described in the neostriatum after this manipulation including a 12–19% decrease in the density of dendritic spines on the medium sized spiny neurons (Ingham et al., 1993; Ingham et al., 1989). Sixty percent of dopaminergic synaptic boutons identified by tyrosine hydroxylase immunoreactivity contact the dendritic spines of these neurons but the synapses are small, symmetric and contact the spine necks (Freund et al., 1984). The main synaptic input to dendritic spines is from the cortex and thalamus, it is thought to be excitatory and the synaptic contacts are large, asymmetric and are often complex in shape (Somogyi et al., 1981; Kemp and Powell, 1971). The decrease in spine density which occurs after removal of the dopaminergic input raises the question of what happens to the excitatory input onto the heads of spines. These synapses may move to an alternative postsynaptic target or disappear after the 6-OHDA lesion. The vast majority of asymmetric synapses in the neostriatum are axo-spinous and so this question can be addressed by examining the numerical density of asymmetric synaptic contacts in the structure after unilateral 6-OHDA lesions.

The present study compared the ultrastructural morphology of enkephalin-immunoreactive boutons and their postsynaptic targets in different territories of the nucleus accumbens in the rat. The synaptic bouton profiles were identified by antibodies directed against [leu5]enkephalin. Ninety-five percent of the synaptic contacts were symmetric in configuration and the remaining 5% were asymmetric. Axosomatic contacts comprised 6% of all enkephalin-immunoreactive junctions and were distributed equally in all parts of the nucleus. Most (76%) synaptic terminals contacted dendrites but they contacted proportionally fewer dendrites in the shell (71%) than in the core (78%). Moreover, enkephalin-immunoreactive synaptic boutons in the shell (19%) and caudal enkephalin-rich areas (17%) of the core contacted twice as many spines than in the remaining parts of the core (8.5%). In the core, long pallidum-like dendrites were occasionally found ensheathed in enkephalin-immunoreactive terminal boutons. We conclude that the differential arrangement of enkephalinergic contacts in the shell and core could have important functional consequences, especially when considered in relation to other known morphological and neurochemical differences between these regions.

Destruction of the dopamine-containing neurons in the rat substantia nigra results in morphological changes in the striatum which have been characterized at both the light and electron microscopic levels. After a unilateral 6-hydroxydopamine injection into the medial forebrain bundle, Golgi-impregnated medium-sized spiny neurons in the neostriatum ipsilateral to the injection had a lower density of spines on their dendrites than those on the contralateral side. A similar decrease in spine density was apparent from 12 days until at least 13.5 months after the lesion. A bilateral loss of spines occurred with increasing age regardless of the presence or absence of the nigrostriatal dopaminergic pathway. At the ultrastructural level, the general pattern of synaptic input to the Golgi-impregnated medium-sized spiny neurons was similar on both sides of the brain. The most obvious class of afferent boutons contacting these spiny neurons formed prominent asymmetrical synaptic specializations with the heads of the spines. The numbers of asymmetric synaptic profiles counted in random electron micrographs from the striata ipsilateral and contralateral to the lesion were not significantly different from each other. A small but significant increase in the length of asymmetric synaptic specialization profiles was, however, detected in the striata lacking a dopamine input.

Dendrites bristling with small protuberances (spines from spina, the Latin word for a thorn) are the characteristic of many classes of neurones in the central nervous system and are best observed in Golgi-impregnated material or in cells filled with other intracellular markers. In the neostriatum the principal output neurones are densely spiny. The principal significance of these thorny structures has intrigued neuroscientists since Cajal. What the importance of those thorny dendrites is. Whether the structure as solid as it looks in the picture or do spines change their shape. How it is possible to explore the properties of these strange structures. Mathematical models suggest that they may be capable of controlling the effectiveness of individual synaptic inputs but the idea is hard to test with current techniques. Recent correlated light and electron microscopic studies in Edinburgh have increased the interest in the neurobiology of spines. Interest grew from the results of experiments designed to help to understand the relevance of dopamine for human medicine.

The anatomical findings and the implications arising from the data presented in this chapter can be summarized as follows. (1) They demonstrate the existence of a direct synaptic input from the globus pallidus to the neurones in the output stations of the basal ganglia. (2) They show that this input is strategically placed in the proximal regions of the neurones, such that it is ideally situated to have a prominent control or influence over the output of the basal ganglia. (3) They suggest that this control or influence is likely to be inhibitory since the terminals derived from the globus pallidus contain, and presumably use, GABA as a transmitter. (4) They demonstrate that the neurones in the output stations of the basal ganglia (some of which have been identified as projection neurones) receive convergent synaptic input from the striatum and the globus pallidus. (5) It is likely that administration of PHA-L to the globus pallidus results in the labelling of subthalamic terminals in the SNr and entopeduncular nucleus. These terminals display glutamate immunoreactivity and converge onto the same neurones that receive input from the striatum and the globus pallidus. (6) The pallidal input to neurones in the substantia nigra at least, and the convergence with striatal terminals appears to be a general phenomenon as deposits of tracers in different regions of the striatum and globus pallidus gave rise to similar patterns of synaptic innervation in the substantia nigra. (7) The present results emphasise the similarities at the synaptic level, between the two major output structures of the basal ganglia, the substantia nigra pars reticulata and the entopeduncular nucleus. (8) The findings summarized in the chapter also provide an antomical substrate for the physiological observations demonstrating that the output neurones of the basal ganglia respond to striatal stimulation by a decrease, increase or a combination of decrease/ increase of their firing rates.

The pattern of enkephalin immunoreactivity was examined in the adult rat neostriatum, at various times after unilateral removal of the nigrostriatal dopamine input by 6-hydroxydopamine injection into the medial forebrain bundle. Animals were examined 12 days, 26 days or 13 months after the lesion. Enkephalin-immunoreactive synaptic boutons (n = 1018) in the control and the dopamine-depleted neostriatum were analysed in the electron microscope. The area of enkephalin-immunoreactive synaptic bouton profiles was significantly larger in the dopamine-depleted neostriatum and this increase was maximal in rats in which the lesion had been made 26 days or 13 months previously (50% increase). The synaptic specializations of these enkephalin-immunoreactive boutons were significantly longer in the neostriatum from the injected side. Dendritic shafts were the principal postsynaptic target of these boutons (67%) but dendritic spines (18%), perikarya (6.5%) and unidentifiable small dendrites or spines (8.5%) were also contacted. The proportions of enkephalin-immunoreactive boutons on the different postsynaptic targets were not altered by the 6-hydroxydopamine lesion. The increase in enkephalin immunoreactivity observed in the dopamine-depleted neostriatum in previous studies may be explained by the increase in the size of enkephalin-immunoreactive synaptic boutons found in the present ultrastructural investigation. The observations do not rule out the possibility that there is also an increase in the number of immunoreactive synaptic boutons, due to, for example, sprouting of the existing enkephalin-containing fibres.