Modulation of synaptic zinc in barrel cortex by whisker stimulation

The cortical representation of the body surface is not fixed, but rather, is continuously modified by ongoing changes in sensory experience. Although the cellular and molecular mechanisms that subserve these changes are uncertain, increasing evidence suggests that synaptically-released zinc may play a role. Zinc is released from a subset of glutamatergic neurons and can modulate postsynaptic excitability by regulating the activation of glutamate and GABA receptor-gated ion channels. Previously, we have shown that whisker plucking, a manipulation commonly used to induce cortical map plasticity, results in a rapid and robust increase in staining levels for synaptic zinc in deprived regions of the barrel cortex. In the present study, we examined the effect of increased whisker activity, analogous to what may happen during tactile learning or exploratory behavior in a natural setting, on synaptic zinc levels in the adult barrel cortex. Our results indicate that stimulation of whiskers caused a selective decrease in zinc levels within layer 4 of the barrel hollow corresponding to the stimulated whisker. Quantitatively, levels of staining were significantly reduced at 3 h, and showed even greater reductions following 12 and 24 h of stimulation. However, these changes were not long-lasting, as levels of staining in the stimulated barrel returned to control values within 24 h after stimulation had ceased. These data indicate that zincergic circuits are highly sensitive to ongoing changes in sensory experience and may participate in moment-to-moment changes in the functional connectivity of the cerebral cortex.     

Differential response of synaptic zinc levels to sensory deprivation in the barrel cortex of young and adult mice

The distribution of synaptic zinc after short-term (up to 48 h) tactile deprivation of vibrissae was investigated in the barrel cortex of mice using histochemical staining. In adult mice, 12 h after trimming selected rows of vibrissae, an increase in zinc staining in the deprived barrels was observed. This increase was still present 48 h after trimming. These results indicate that the level of synaptic zinc is rapidly regulated by neuronal activity in adult mice. In young (8-day-old) mice, the short-term deprivation did not alter zinc staining and only chronic sensory deprivation produced an increase in zinc staining. However, after long-term deprivation no changes were found in adult mice. These results suggest that different mechanisms might be involved in functional reorganization of zinc containing terminals in young and fully mature cerebral cortex.     

Passive avoidance performance of mice fed marginally or severely zinc deficient diets during post-embryonic brain development

Swiss-Webster outbred mice were fed marginally or severely zinc deficient diets (9 or 2 ppm zinc) from day 16 gestation to day 15 postnatal. Control mice were fed a 100 ppm diet either ad lib or in amounts equal to the diet intake of deprived mice (pair fed controls). Male and female offspring were tested at 70 days of age in a one-trial passive avoidance task with a 30 min train-test interval. Both marginally and severely zinc deprived offspring (but not pair fed controls) had shorter avoidance latencies than offspring of ad lib fed zinc replete controls. Zinc deprived offspring did not differ from control mice when either baseline or “stressed” (exposure to novel environment) plasma corticosterone levels were quantitated. Further, zinc staining patterns of the hippocampus (Timm-sulfide stain) were not altered in the nutritionally deprived offspring. Thus marginal dietary zinc deficiency during development can lead to impaired passive avoidance performance in adult mice. This behavioral effect is not associated with altered pituitary-adrenocortical activity or with a permanent reduction in hippocampal zinc staining. This result has significant implications for the influence of zinc deprivation in utero and in the neonatal animal on adult behavior characteristics.     

Zinc histochemistry reveals circuit refinement and distinguishes visual areas in the developing ferret cerebral cortex

A critical question in brain development is whether different brain circuits mature concurrently or with different timescales. To characterize the anatomical and functional development of different visual cortical areas, one must be able to distinguish these areas. Here, we show that zinc histochemistry, which reveals a subset of glutamatergic processes, can be used to reliably distinguish visual areas in juvenile and adult ferret cerebral cortex, and that the postnatal decline in levels of synaptic zinc follows a broadly similar developmental trajectory in multiple areas of ferret visual cortex. Zinc staining in all areas examined (17, 18, 19, 21, and Suprasylvian) is greater in the 5-week-old than in the adult. Furthermore, there is less laminar variation in zinc staining in the 5-week-old visual cortex than in the adult. Despite differences in staining intensity, areal boundaries can be discerned in the juvenile as in the adult. By 6 weeks of age, we observe a significant decline in visual cortical synaptic zinc; this decline was most pronounced in layer IV of areas 17 and 18, with much less change in higher-order extrastriate areas during the important period in visual cortical development following eye opening. By 10 weeks of age, the laminar pattern of zinc staining in all visual areas is essentially adultlike. The decline in synaptic zinc in the supra- and infragranular layers in all areas proceeds at the same rate, though the decline in layer IV does not. These results suggest that the timecourse of synaptic zinc decline is lamina specific, and further confirm and extend the notion that at least some aspects of cortical maturation follow a similar developmental timecourse in multiple areas. The postnatal decline in synaptic zinc we observe during the second postnatal month begins after eye opening, consistent with evidence that synaptic zinc is regulated by sensory experience.     

Zincergic innervation of the forebrain distinguishes epilepsy-prone from epilepsy-resistant rat strains

Zinc is released from a subset of cerebral cortical neurons whereupon it exerts a powerful modulatory influence on excitatory and inhibitory neurotransmission. A number of studies have suggested that alterations in the regulation of zinc may contribute to the genesis of epilepsy. Here, we tested this hypothesis by examining the distribution of zinc-containing axon terminals in rats selectively bred for an innate susceptibility (FAST) or resistance (SLOW) to the development of kindling-induced seizures. Zinc was stained histochemically and levels of staining were quantitatively assessed. We found that the levels of synaptic zinc were significantly lower in the SLOW rats throughout the telencephalon. This relative reduction was most pronounced in limbic cortices where levels were less than 30% of FAST rats. These results suggest that innate differences in the homeostatic regulation of synaptic zinc, particularly in limbic cortices, may underlie differences in epileptogenicity.     

Changes in expression of a synaptic vesicle antigen in aging sympathetic neurons

The effects of altering synaptic activity of sympathetic neurons on the expression of a synaptic vesicle protein (p65) were studied by deafferentation of the superior cervical ganglion (SCG) in adult and aged Fischer-344 rats. Levels of p65, an integral membrane protein of synaptic vesicles, were assayed by radioimmunoassay. After deafferentation, a transient increase in p65 levels is observed in the SCG of adult rats. In aged animals, the response to deafferentation is delayed and enhanced, and levels do not drop to values observed in operated adults. After SCG deafferentation, p65 levels in the iris, an SCG target, initially are depressed below control levels; p65 levels return to control values in adult animals after 14 days, but remain depressed in aged animals. In contrast, a transient increase in p65 levels is observed in the pineal of both adult and aged animals. These results suggest that while the aged sympathetic nervous system retains the ability to respond to alterations in synaptic activity, it is unable to reregulate once a response is initiated.     

Altered maturation of the primary somatosensory cortex in a mouse model of fragile X syndrome

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and results from the loss of the fragile X mental retardation protein (FMRP). Many fragile X-related cognitive and behavioral features emerge during childhood and are associated with abnormal synaptic and cellular organization of the cerebral cortex. Identifying the roles of FMRP in cortical development will provide a basis for understanding the pathogenesis of the syndrome. However, how the loss of FMRP influences the developmental trajectory of cortical maturation remains unclear. We took advantage of the stereotyped and well-characterized development of the murine primary somatosensory cortex to examine cortical maturation during a time-window that corresponds to late embryonic and early postnatal development in the human. In the Fmr1 knockout mouse, we find a delay in somatosensory map formation, alterations in the morphology profile of dendrites and spines of layer 4 neurons and a decrease in the synaptic levels of proteins involved in glutamate receptor signaling at times corresponding to the highest levels of FMRP expression. In contrast, cortical arealization, synaptic density in layer 4 and early postnatal regulation of mRNAs encoding synaptic proteins are not altered in Fmr1 knockout mice. The specificity of the developmental delay in Fmr1 knockout mice indicates that the loss of FMRP does not result in a general stalling of cerebral cortex maturation. Instead, our results suggest that inaccurate timing of developmental processes caused by the loss of FMRP may lead to alterations in neural circuitry that underlie behavioral and cognitive dysfunctions associated with FXS.     

Histochemical changes in enzymes of energy metabolism in the dentate gyrus accompany deafferentation and synaptic reorganization

The dentate gyrus of adult rats was examined histochemically for cytochrome oxidase and lactate dehydrogenase activity after unilateral lesions of the entorhinal cortex. In normal animals, synaptic terminal fields of the perforant pathway from the entorhinal cortex show high levels of cytochrome oxidase activity (the other two-thirds dentate molecular layer), whereas terminal zones of the commissural and associational fibers show high levels of lactate dehydrogenase activity (the inner one-third dentate molecular layer). Lesions of the entorhinal cortex result in a significant reduction in staining for cytochrome oxidase in the deafferented outer molecular layer of the dentate gyrus. The changes become prominent at 16-24 h after the lesion and persist until 90 days, the longest post-lesion survival time studied. In the non-deafferented inner zones ipsilateral to the lesion, there is an increase in staining for cytochrome oxidase and lactate dehydrogenase at 24 h post-lesion that disappears by days 2-4. From 8 to 90 days post-lesion, the band of high reactivity for lactate dehydrogenase in the inner molecular layer spreads approximately 40 microns into the overlying deafferented zone. This expansion parallels the expansion of the commissural and associational terminal fields into the adjacent deafferented molecular layer. Thus, lesion-induced synaptogenesis in the dentate gyrus is accompanied by a corresponding change in enzyme activity. The results indicate that the pattern of activity of enzymes involved in energy metabolism in the dentate gyrus depends on the distribution of pathway-specific synaptic input.     

Supplemental zinc restores antibody formation in cultures of aged spleen cells. III. Impairment of II-2-mediated responses

The effects of zinc on interleukin-2(IL-2)-dependent T cell responses of lymphocytes from immunodepressed aged mice and from young adult animals were studied. Concentrations of zinc which have been shown to restore antibody formation in cells from aged mice and to increase the production of Il-1 and Il-4 inhibited the production of Il-2. Cells from both young adults and aged mice were inhibited similarly. Zinc also impaired the ability of aged T cells to proliferate in response to concanavalin A and exogenous Il-2, but enhanced the proliferation of similarly activated splenic cultures containing both T and B cells. Cultures of isolated B lymphocytes produced antibody to sheep red blood cells if the cells were provided with supplemental zinc and Il-1. In contrast, recombinant Il-2 with or without zinc did not activate antibody formation. The results support the premise that the restorative effects of zinc are independent of Il-2 and that Il-2 is not a necessary mediator for antibody production in the aged.     

Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development

The rat retrosplenial cortex is part of a heavily interconnected limbic circuit, considered to have an important role in spatial memory. Interestingly, the granular retrosplenial cortex has an exceptionally distinct system of dendritic bundles, originating from callosally projecting pyramidal neurons in layer II. These can be detected as early as postnatal day 5; and, although their functional significance remains to be elucidated, the existence of these bundles makes the granular retrosplenial cortex an attractive model system for a wide range of development and functional investigations. Here, we report four results concerning the development of modularity in the granular retrosplenial cortex in rats as investigated by neurochemical markers associated to cortico-cortical and thalamo-cortical connections. Emphasis is placed on zinc, an activity-related substance associated with glutamatergic, non-thalamic terminations. 1) Zinc shows a transient strong expression during early postnatal development, but later than the appearance of the upper layer bundles (at postnatal day 5). By postnatal day 11 to postnatal day 15 staining for zinc achieved its most complex pattern; such that layer I had an elaborate organization both in the tangential and radial dimensions. Three sublaminae were distinguished (layers Ia-c): a superficial, thin tier (Ia) with patchy, moderate staining which periodically intruded into the underlying layer Ib ("funnel" modules), a middle band of variable width and light staining (Ib), and a deep, thin band with heavy and patchy staining (Ic) which, at rostral levels, spread upward into layer Ib (as "dome-like" modules). 2) At postnatal day 15, immunohistochemical methods showed that layers Ia, b zinc-funnels were co-localized with glutamate receptor subunits 2/3, GABA receptor type A alpha1 subunit and the thalamo-cortical marker, vesicular glutamate transporter 2. Layer Ic and the zinc dome-like modules were co-labeled for the cortico-cortical marker, vesicular glutamate transporter 1 and calretinin. 3) The spatial coincidence between zinc funnels in layers Ia, b and vesicular glutamate transporter 2 was further investigated by electron microscopy, which demonstrated co-localization of zinc and vesicular glutamate transporter 2 in synaptic boutons. The unusual co-localization of zinc and thalamo-cortical terminations was confirmed by retrograde transport of zinc to neurones in the anterodorsal thalamic nucleus at postnatal day 9 and postnatal day 13, and can thus be considered a transient zinc expression in thalamo-cortical boutons. This was not observed at postnatal day 28 or later. 4) After postnatal day 18, zinc staining started to fade in all layers. Before postnatal day 21, the heavy staining for zinc in the domes had completely disappeared. Zinc staining in layer Ia and the funnels virtually disappeared after postnatal day 28. A transient expression of zinc is reported in at least one other cortical area (layer IV of barrel cortex from postnatal day 5 to postnatal day 14, maximal at postnatal days 9-11). We conclude that the transient expression of zinc can occur in both limbic and sensory areas, and that down-regulation of zinc in cortical modules might be related to synaptic plasticity and remodeling during development.     

Copper, manganese and zinc in the developing brain of control and quaking mice

Copper, manganese and zinc were measured by flameless atomic absorption spectrophotometry in the developing brain of normal and quaking mice. The latter is a neurological mutant presenting early arrest of myelination. Copper concentration was increased by 200% between 10 and 20 days after birth and then leveled off in adult mice. Manganese concentration increased both in control mice and in quaking mice from 3 to 20 days by 200% and then decreased by 19% in control mice and 24% in quaking mice at adult age. Zinc increased by 93% in control and 173% in quaking mice between 10 and 20 days of age, and then progressively declined until 62 days. The mouse brain accumulates considerably all the 3 metals during early development. During the first 20 days, the augmentation is 6-fold for copper, 5-fold for manganese and 5.5-fold for zinc. In quaking, alterations are not very important.     

Massive restructuring of neuronal circuits during functional reorganization of adult visual cortex

The cerebral cortex has the ability to adapt to altered sensory inputs. In the visual cortex, a small lesion to the retina causes the deprived cortical region to become responsive to adjacent parts of the visual field. This extensive topographic remapping is assumed to be mediated by the rewiring of intracortical connections, but the dynamics of this reorganization process remain unknown. We used repeated intrinsic signal and two-photon imaging to monitor functional and structural alterations in adult mouse visual cortex over a period of months following a retinal lesion. The rate at which dendritic spines were lost and gained increased threefold after a small retinal lesion, leading to an almost complete replacement of spines in the deafferented cortex within 2 months. Because this massive remodeling of synaptic structures did not occur when all visual input was removed, it likely reflects the activity-dependent establishment of new cortical circuits that serve the recovery of visual responses.     

Zinc chelation during non-lesioning overexcitation results in neuronal death in the mouse hippocampus

In the hippocampus, chelatable zinc is accumulated in vesicles of glutamatergic presynaptic terminals, abounding specially in the mossy fibers, from where it is released with activity and can exert a powerful inhibitory action upon N-methyl-D-aspartate receptors. Zinc is therefore in a strategic situation to control overexcitation at the zinc-rich excitatory synapses, and consequently zinc removal during high activity might result in excitotoxic neuronal damage. We analyzed the effect of zinc chelation with sodium dietyldithiocarbamate under overexcitation conditions induced by non-lesioning doses of kainic acid in the mouse hippocampus, to get insight into the role of zinc under overexcitation. Swiss male mice were injected with kainic acid (15 mg/kg, i.p.) 15 min prior to sodium dietyldithiocarbamate (150 mg/kg, i.p.), and left to survive for 6 h, 1 day, 4 days, or 7 days after the treatment. Cell damage was analyzed with the hematoxylin-eosin and acid fuchsin stainings. Neither control animals treated only with kainic acid nor those treated only with sodium dietyldithiocarbamate suffered seizures or neuronal damage. By contrast, the kainic acid+sodium dietyldithiocarbamate-treated animals showed convulsive behavior and cell death involving the hilus, CA3, and CA1 regions. Pretreatment with the N-methyl-D-aspartate receptor antagonist MK801 (1 mg/kg, i.p.) completely prevented neuronal damage. Experiments combining different doses of sodium dietyldithiocarbamate and kainic acid with different administration schedules demonstrated that the overlap of zinc chelation and overexcitation is necessary to trigger the observed effects. Moreover, the treatment with a high dose of sodium dietyldithiocarbamate (1000 mg/kg), which produced a complete bleaching of the Timm staining for approximately 12 h, highly increased the sensitivity of animals to kainic acid. Altogether, our results indicate that the actions of sodium dietyldithiocarbamate are based on a reduction of zinc levels, which under overexcitation conditions induce seizures and neuronal damage. These findings fully support a protective role for synaptically released zinc during high neuronal activity, most probably mediated by its inhibitory actions on N-methyl-D-aspartate receptors, and argue against a direct action of synaptic zinc on the observed neuronal damage.     

Continuous enriched environment improves learning and memory in adult NMRI mice through theta burst-related-LTP independent mechanisms but is not efficient in advanced aged animals

Introduction

Effects of 3-month continuous environmental enrichment (EE) on cognitive abilities and on theta burst-related synaptic plasticity of CA1 hippocampal neuronal networks have been assessed in 6- and 20-month old NMRI female mice.

Results

EE decreased anxiety-like behavior and improved learning and memory performances in adult but not in aged mice. Electrophysiological results in CA1 hippocampal slices showed that basal synaptic transmission was not affected by EE in adult mice whereas it was partially improved in aged animals, even though not sufficient to rescue the decrease related to aging. Besides, no effect of EE on N-methyl-d-aspartate receptor activation and theta-burst-induced long-term potentiation was found in adult or aged animals.

Discussion

These results indicate that continuous EE is able to improve cognitive abilities in adult NMRI female mice, that does not correlate with changes in theta burst-related synaptic plasticity within neuronal networks. In addition, the lack of effects in aged animals suggests the existence of a critical delay for the beneficial effects of EE on cognitive aging.     

Enhancement of cortical plasticity by behavioral training in acetylcholine-depleted adult rats

Trimming all whiskers except two on one side of an adult rat's face results in cortical plasticity in which the spared whiskers, D2 and one D-row surround whisker (either D1 or D3), evoked responses containing more spikes than the response evoked by the cut whisker (called whisker pairing plasticity). Previously we have reported that acetylcholine (ACh) depletion in cortex prevents surround D-row whisker plasticity from developing within the barrel cortex. In this study we examined whether the animal's active use of its two intact whiskers can restore some aspects of plasticity in the ACh-depleted cortex. To achieve this goal, ACh was depleted from barrel field cortex, and 14 days after the depletion surgery, whiskers were trimmed and animals were trained on a whisker-dependent gap crossing task. After 7 days of training, animals were anesthetized with urethan and prepared for single-unit recording. Training the ACh-depleted, whisker-paired animals resulted in a significant enhancement of responses to paired surround whiskers: the D-paired whisker-evoked response contained more spikes than the D-cut evoked response. We conclude that training whisker paired rats has a positive impact on response properties of neurons in S1 cortex, even in ACh-depleted animals.     

小鼠脑中Zn元素和ZnT3 mRNA表达的研究

目的：研究ZnT3 mRNA的表达与Zn等金属元素在脑中精细分布的相互作用和功能。方法:使用同步辐射X射线荧光法（SRXRF）测定小鼠全脑和脑切片中Zn等金属元素分布，同时使用反转录多聚酶链式反应（RT-PCR）检测小鼠各组织中的ZnT3 mRNA的表达量。结果:脑中Zn元素不是均匀分布的，主要分布在皮层、海马和齿状回部位。大脑皮层、海马和睾丸中的ZnT3 mRNA有较高丰度，而其它组织中未检出ZnT3 mRNA 。结论：ZnT3能促进胞浆内Zn富集于囊泡中，通过介导胞浆锌的跨膜转运过程，构造囊泡“锌池”。     

Experience-dependent regulation of vesicular zinc in male and female 3xTg-AD mice

In the mammalian cerebral cortex, zinc is an important modulator of synaptic transmission and conversely, plasticity. Zinc is also involved, in a sex-dependent manner, in the pathogenesis of Alzheimer's disease (AD), where substantial declines in plasticity may occur. To examine this relationship further, the regulation of vesicular zinc was examined after the induction of cortical plasticity through vibrissae plucking in male and female C57Bl/6 and 3xTg-AD mice at various age points. Female C57Bl/6 mice were found to have an elevated response compared to male C57Bl/6 mice through mid-adult ages, a sex-difference likely mediated by the differential regulation of vesicular zinc by the sex hormones. Male 3xTg-AD mice had a significantly greater zincergic response compared to C57Bl/6 mice, which is likely indicative of a compensatory mechanism utilized by the male 3xTg-AD mice to combat the decline in plasticity associated with the AD state. These results exemplify how the regulation of vesicular zinc may be a significant component in the progression of AD, especially regarding the sex-dependent element.     

Extracellular chelation of zinc does not affect hippocampal excitability and seizure-induced cell death in rats

In the nervous system, zinc can influence synaptic responses and at extreme concentrations contributes to epileptic and ischaemic neuronal injury. Zinc can originate from synaptic vesicles, the extracellular space and from intracellular stores. In this study, we aimed to determine which of these zinc pools is responsible for the increased hippocampal excitability observed in zinc-depleted animals or following zinc chelation. Also, we investigated the source of intracellularly accumulating zinc in vulnerable neurons. Our data show that membrane-permeable and membrane-impermeable zinc chelators had little or no effect on seizure activity in the CA3 region. Furthermore, extracellular zinc chelation could not prevent the accumulation of lethal concentrations of zinc in dying neurons following epileptic seizures. At the electron microscopic level, zinc staining significantly increased at the presynaptic membrane of mossy fibre terminals in kainic acid-treated animals. These data indicate that intracellular but not extracellular zinc chelators could influence neuronal excitability and seizure-induced zinc accumulation observed in the cytosol of vulnerable neurons.     

Short exposure to an enriched environment accelerates plasticity in the barrel cortex of adult rats

Cortical sensory neurons adapt their response properties to use and disuse of peripheral receptors in their receptive field. Changes in synaptic strength can be generated in cortex by simply altering the balance of input activity, so that a persistent bias in activity levels modifies cortical receptive field properties. Such activity-dependent plasticity in cortical cell responses occurs in rat cortex when all but two whiskers are trimmed for a period of time at any age. The up-regulation of evoked responses to the intact whiskers is first seen within 24 h in the supragranular layers [Laminar comparison of somatosensory cortical plasticity. Science 265(5180):1885-1888] and continues until a new stable state is achieved [Experience-dependent plasticity in adult rat barrel cortex. Proc Natl Acad Sci U S A 90(5):2082-2086; Armstrong-James M, Diamond ME, Ebner FF (1994) An innocuous bias in whisker use in adult rat modifies receptive fields of barrel cortex neurons. J Neurosci 14:6978-6991]. These and many other results suggest that activity-dependent changes in cortical cell responses have an accumulation threshold that can be achieved more quickly by increasing the spike rate arising from the active region of the receptive field. Here we test the hypothesis that the rate of neuronal response change can be accelerated by placing the animals in a high activity environment after whisker trimming. Test stimuli reveal an highly significant receptive field bias in response to intact and trimmed whiskers in layer IV as well as in layers II-III neurons in only 15 h after whisker trimming. Layer IV barrel cells fail to show plasticity after 15-24 h in a standard cage environment, but produce a response bias when activity is elevated by the enriched environment. We conclude that elevated activity achieves the threshold for response modification more quickly, and this, in turn, accelerates the rate of receptive field plasticity.     

Age-related changes in sulfide-silver staining in the rat neostriatum: a quantitative histochemical study.

The density and distribution of sulfide-silver staining in the neostriatum of 3-, 12-, and 24-month-old male Sprague-Dawley rats were analyzed using the neo-Timm sulfide-silver histochemical technique associated with microdensitometry. This technique stains zinc-containing terminals in the striatum and the density of neo-Timm staining is considered to be parallel to the density of synaptic boutons containing zinc. In the neostriatum sulfide-silver, staining was intense in the matrix, although the striosomes did not show appreciable reactivity. The density of sulfide-silver staining was significantly reduced (p less than 0.001) in the matrix of 12-month-old in comparison to 3-month-old rats. No further changes were noticeable between 24- and 12-month-old rats. In contrast, the area and the perimeter of neostriatum that were assessed by quantitative image analysis did not show age-related changes. The present results indicated that similar to the observations for a variety of neurochemical parameters of rat neostriatum such as local cerebral glucose utilization, cholinergic muscarinic receptors, and dopamine D-1 receptors, zinc-containing striatal terminal were primarily decreased between young and adult subjects but not between adult and aged animals.