Unlabeled Spines Research Articles

Synaptic distributions of GluA2 and PKMζ in the monkey dentate gyrus and their relationships with aging and memory.

Rhesus monkeys provide a valuable model for studying the neurobiological basis of cognitive aging, because they are vulnerable to age-related memory decline in a manner similar to humans. In this study, young and aged monkeys were first tested on a well characterized recognition memory test (delayed nonmatching-to-sample; DNMS). Then, electron microscopic immunocytochemistry was performed to determine the subcellular localization of two proteins in the hippocampal dentate gyrus (DG): the GluA2 subunit of the glutamate AMPA receptor and the atypical protein kinase C ζ isoform (PKMζ). PKMζ promotes memory storage by regulating GluA2-containing AMPA receptor trafficking. Thus, we examined whether the distribution of GluA2 and PKMζ is altered with aging in DG axospinous synapses and whether it is coupled with memory deficits. Monkeys with faster DNMS task acquisition and more accurate recognition memory exhibited higher proportions of dendritic spines coexpressing GluA2 and PKMζ. These double-labeled spines had larger synapses, as measured by postsynaptic density area, than single-labeled and unlabeled spines. Within this population of double-labeled spines, aged monkeys compared with young expressed a lower density of synaptic GluA2 immunogold labeling, which correlated with lower recognition accuracy. Additionally, higher density of synaptic PKMζ labeling in double-labeled spines correlated with both faster task acquisition and better retention. Together, these findings suggest that age-related impairment in maintenance of GluA2 at the synapse in the primate hippocampus is coupled with memory deficits.

Vesicular glutamate transporter 1 and vesicular glutamate transporter 2 synapses on cholinergic neurons in the sublenticular gray of the rat basal forebrain: a double-label electron microscopic study

The basal forebrain (BF) comprises morphologically and functionally heterogeneous cell populations, including cholinergic and non-cholinergic corticopetal neurons that are implicated in sleep–wake modulation, learning, memory and attention. Several studies suggest that glutamate may be among inputs affecting cholinergic corticopetal neurons but such inputs have not been demonstrated unequivocally. We examined glutamatergic axon terminals in the sublenticular substantia innominata in rats using double-immunolabeling for vesicular glutamate transporters (Vglut1 and Vglut2) and choline acetyltransferase (ChAT) at the electron microscopic level. In a total surface area of 30,000 μm2, we classified the pre- and postsynaptic elements of 813 synaptic boutons. Vglut1 and Vglut2 boutons synapsed with cholinergic dendrites, and occasionally Vglut2 axon terminals also synapsed with cholinergic cell bodies. Vglut1 terminals formed synapses with unlabeled dendrites and spines with equal frequency, while Vglut2 boutons were mainly in synaptic contact with unlabeled dendritic shafts and occasionally with unlabeled spines. In general, Vglut1 boutons contacted more distal dendritic compartments than Vglut2 boutons. About 21% of all synaptic boutons (n=347) detected in tissue that was stained for Vglut1 and ChAT were positive for Vglut1, and 14% of the Vglut1 synapses were made on cholinergic profiles. From separate cases stained for Vglut2 and ChAT, 35% of all synaptic boutons (n=466) were positive for Vglut2, and 23% of the Vglut2 synapses were made on cholinergic profiles. On average, Vglut1 boutons were significantly smaller than Vglut2 synaptic boutons. The Vglut2 boutons that synapsed cholinergic profiles tended to be larger than the Vglut2 boutons that contacted unlabeled, non-cholinergic postsynaptic profiles. The presence of two different subtypes of Vgluts, the size differences of the Vglut synaptic boutons, and their preference for different postsynaptic targets suggest that the action of glutamate on BF neurons is complex and may arise from multiple afferent sources.

Ultrastructural and metabolic changes in the neuropeptide Y‐containing striatal neuronal network after thermocoagulatory cortical lesion in adult rat

This study examined the effects of unilateral thermocoagulatory cortical lesion on the pattern of neuropeptide Y immunostaining in the rat ipsilateral striatum at 4 and 21 days post-lesion. Light microscopic analysis showed a significant increase in the number of neuropeptide Y-positive neurons vs. control at both time points; paradoxically, the intraneuronal level of labelling significantly decreased at 4 days post-lesion but increased at 21 days post-lesion. Ultrastructural analysis in control condition showed a higher proportion of dendritic versus axonal labelled processes (3.5 ratio); all the neuropeptide Y synaptic terminals formed symmetrical contacts, mostly onto unlabelled dendrites. At 4 days post-lesion, the neuropeptide Y-positive axon density dramatically increased (+576%) without significant change in the labelled dendrite density, vs. control values; the density of neuropeptide Y synaptic terminals increased in parallel by 233%. In addition, a significant proportion of large neuropeptide Y boutons forming asymmetrical synapses onto unlabelled spines were observed. At 21 days post-lesion, densities of neuropeptide Y dendrites, axons, and synaptic terminals increased by 68, 246 and 125%, respectively, vs. control. But, the morphological features of the neuropeptide Y axonal processes and synaptic specializations of the boutons were similar to those observed in control condition. These data (1) raise an important issue regarding the origin of the terminals forming asymmetrical synapses in the striatum, (2) suggest that adaptative changes in the neuropeptide Y neuronal network may be a main component of striatal remodelling resulting from the progressive loss of cortical inputs, and (3) reinforce the view that neuropeptide Y and excitatory amino acid functions may be tightly linked in the striatum. Synapse 34:208–221, 1999. © 1999 Wiley-Liss, Inc.

Ultrastructural and metabolic changes in the neuropeptide Y-containing striatal neuronal network after thermocoagulatory cortical lesion in adult rat.

This study examined the effects of unilateral thermocoagulatory cortical lesion on the pattern of neuropeptide Y immunostaining in the rat ipsilateral striatum at 4 and 21 days post-lesion. Light microscopic analysis showed a significant increase in the number of neuropeptide Y-positive neurons vs. control at both time points; paradoxically, the intraneuronal level of labelling significantly decreased at 4 days post-lesion but increased at 21 days post-lesion. Ultrastructural analysis in control condition showed a higher proportion of dendritic versus axonal labelled processes (3.5 ratio); all the neuropeptide Y synaptic terminals formed symmetrical contacts, mostly onto unlabelled dendrites. At 4 days post-lesion, the neuropeptide Y-positive axon density dramatically increased (+576%) without significant change in the labelled dendrite density, vs. control values; the density of neuropeptide Y synaptic terminals increased in parallel by 233%. In addition, a significant proportion of large neuropeptide Y boutons forming asymmetrical synapses onto unlabelled spines were observed. At 21 days post-lesion, densities of neuropeptide Y dendrites, axons, and synaptic terminals increased by 68, 246 and 125%, respectively, vs. control. But, the morphological features of the neuropeptide Y axonal processes and synaptic specializations of the boutons were similar to those observed in control condition. These data (1) raise an important issue regarding the origin of the terminals forming asymmetrical synapses in the striatum, (2) suggest that adaptative changes in the neuropeptide Y neuronal network may be a main component of striatal remodelling resulting from the progressive loss of cortical inputs, and (3) reinforce the view that neuropeptide Y and excitatory amino acid functions may be tightly linked in the striatum.

Thalamic input to parvalbumin-immunoreactive GABAergic interneurons: organization in normal striatum and effect of neonatal decortication

The neocortex and thalamus send dense glutaminergic projections to the neostriatum. The neocortex makes synaptic contact with spines of striatal projection neurons, and also targets a distinct class of GABAergic interneurons immunoreactive for the calcium-binding protein parvalbumin. We determined whether the parafascicular thalamic nucleus also targets striatal parvalbumin-immunoreactive interneurons. The anterograde tracer biotinylated dextranamine was injected into the parafascicular nucleus of adult rats. Double-labeled histochemistry/immunohistochemistry revealed overlapping thalamic fibres and parvalbumin-immunoreactive neurons in the neostriatum. Areas of overlap within the sensorimotor striatum were analysed by electron microscopy. Of 311 synaptic boutons originating from the parafascicular nucleus, 75.9% synapsed with unlabeled dendrites, 22.5% with unlabeled spines, and 1.3% had parvalbumin-immunoreactive dendrites as a postsynaptic target. Only 4% of all asymmetric synapses on parvalbumin-immunoreactive dendrites were derived from the parafascicular nucleus. A separate group of animals underwent bilateral neocortical deafferentation on the third postnatal day, prior to injection of anterograde tracer into the parafascicular nucleus of adult animals. These experiments were performed with the dual purpose of (i) reducing the possibility that thalamic inputs to parvalbumin-immunoreactive neurons are the result of transsynaptic uptake of tracer by a thalamo-cortico-striatal route, and (ii) determining whether competitive interactions between developing corticostriatal and thalamostriatal fibres may account for the relatively sparse thalamic input onto parvalbumin-immunoreactive interneurons. In decorticates, 219 striatal synaptic contacts derived from the parafascicular nucleus, out of which 77.2% were on unlabeled dendrites, 20.9% were upon unlabeled spines, and 0.9% targeted parvalbumin-immunoreactive dendrites. We conclude that the thalamic parafascicular nucleus indeed sends synaptic input to parvalbumin-immunoreactive striatal neurons. Parafascicular nucleus inputs to striatal parvalbumin-immunoreactive interneurons are sparse in comparison to other asymmetric inputs, most of which are likely to be of cortical origin. The synaptic profile of thalamostriatal inputs to parvalbumin-immunoreactive neurons and unlabeled elements is unchanged following neonatal decortication. This suggests that competitive interaction between developing thalamostriatal and corticostriatal projections is not a major mechanism determining synaptic input to striatal subpopulations.

Hippocampal ?2A-adrenergic receptors are located predominantly presynaptically but are also found postsynaptically and in selective astrocytes

Alpha-adrenergic receptor, subtype 2A (alpha2A-AR), activation is one of the primary modes of action for norepinephrine (NE) in the rat hippocampal formation. In this study, alpha2A-AR immunoreactivity (alpha2A-AR-I) was localized by light and electron microscopy in the rat hippocampus and dentate gyrus by using a previously characterized antibody to the rat alpha2A-AR. By light microscopy, dense alpha2A-AR-I was observed in the pyramidal and granule cell layers. Diffuse and slightly granular alpha2A-AR-I was found in the neuropil in all other laminae, notably stratum lacunosum-moleculare. Ultrastructurally, alpha2A-AR-I was found in neuronal cytoplasm associated with large multivesicular-like organelles and with clusters adjacent to endoplasmic reticula and/or plasmalemma. The distribution of alpha2A-AR-I in the strata oriens, radiatum, and lacunosum-moleculare of hippocampal CA1 and CA3 regions and in the molecular layer of the dentate gyrus was remarkably similar (n > 2,000 profiles examined): alpha2A-AR-I was found in axons and terminals (approximately 40%), glia (approximately 30%), dendritic spines (approximately 25%), and dendritic shafts (approximately 5%). This mixed pre- and postsynaptic distribution was not seen in the stratum lucidum of the CA3 region and the dentate hilar region, where most alpha2A-AR-I was found in axons (approximately 60%) and glia (approximately 30%). Alpha-2A-AR-labeled axons were small and unmyelinated; labeled terminals usually formed asymmetric synapses on unlabeled spines; and labeled dendritic spines were morphologically similar to pyramidal or granule cells. Dual labeling studies demonstrated that some axons contained alpha2A-AR-I and tyrosine hydroxylase (TH), the catecholaminergic synthesizing enzyme, and that some TH-labeled terminals were in close proximity to alpha2A-AR-labeled spines and glia. These studies demonstrate that hippocampal alpha2A-AR-I is localized (1) presynaptically in both noncatecholaminergic and catecholaminergic terminals, (2) postsynaptically in the dendritic spines of pyramidal and granule cells near catecholaminergic terminals, and (3) in some glial processes. These results suggest several sites for NE to exert its effects on hippocampal alpha2A-ARs.

Dendritic spines containing ?-opioid receptors in rat striatal patches receive asymmetric synapses from prefrontal corticostriatal afferents

Prefrontal corticostriatal afferents to the caudate-putamen nucleus (CPN) have been implicated in motor and cognitive functions that are subject to opioid modulation through the mu-opioid receptor (MOR). We examined the cellular basis for this modulation by combining anterograde transport of biotinylated dextran amine (BDA) following injections into the rat prefrontal cortex with immunocytochemical detection of MOR in patch compartments of the CPN. The BDA-labeled neurons in deep layer V and layer VI of the dorsal part of the anterior cingulate cortex and the medial agranular cortex projected bilaterally, with an ipsilateral predominance, to MOR-enriched patches in the dorsomedial and dorsocentral CPN, respectively. BDA-labeled terminals often apposed MOR-immunoreactive dendrites and perikarya but formed exclusively asymmetric, excitatory-type synapses mainly with dendritic spines. Of the total anterogradely labeled axon terminals forming asymmetric synapses, 40% (151 of 377) were with MOR-labeled spines, and 58% (220 of 377) were with unlabeled dendritic spines. In addition, immunogold-silver particles for MOR were seen in 14% (134 of 938) of all BDA-labeled axons and axon terminals. These dually labeled axon terminals also formed asymmetric synapses with dendritic spines that contained MOR immunoreactivity. The proportions of BDA-labeled axon terminals forming asymmetric synapses with MOR-labeled or unlabeled spines were similar in the CPN ipsilateral and contralateral to the cortical injections. These results suggest that, in patch compartments of the CPN, MOR plays a critical role in postsynaptic response to, but also in presynaptic modulation of, prefrontal corticostriatal excitation.

Axon terminals immunolabeled for dopamine or tyrosine hydroxylase synapse on GABA-immunoreactive dendrites in rat and monkey cortex.

Dopamine afferents to the cortex regulate the excitability of pyramidal neurons via a direct synaptic input. However, it has not been established whether dopamine also modulates pyramidal cell activity indirectly through synapses on gamma-aminobutyric acid (GABA) interneurons, and whether such inputs differ across cortical regions and species. We sought to address these issues by an immunocytochemical electron microscopic approach that combined peroxidase staining for dopamine or tyrosine hydroxylase (TH) with a pre-embedding gold-silver marker for GABA. In the deep layers of the rat prefrontal cortex and in the superficial layers of the monkey prefrontal and primary motor cortices, terminal varicosities immunoreactive for dopamine or TH formed primarily thin, symmetric synapses on distal dendrites. Both GABA-immunoreactive dendrites as well as unlabeled spines and dendrites were contacted by dopamine- or TH-immunoreactive terminals. Synaptic specializations were detected at some, but not all of these contacts. The relative frequency of these appositional and synaptic contacts did not appear to differ between the rat and monkey prefrontal cortex, or between the monkey prefrontal and motor cortices. Across regions and species, labeled and unlabeled targets of dopamine- or TH-positive terminals received additional synaptic input from unlabeled, and occasionally GABA-immunoreactive terminals. Close appositions between dopamine- or TH-immunoreactive and GABA-positive terminals were observed only rarely. These findings indicate that dopamine afferents provide direct synaptic inputs to GABA local circuit neurons in a consistent fashion across cortical regions and species. Thus, dopamine's cellular actions involve direct as well as modulatory effects on both GABA interneurons and pyramidal projection neurons.

Hippocampal and midline thalamic fibers and terminals in relation to the choline acetyltransferase‐immunoreactive neurons in nucleus accumbens of the rat: A light and electron microscopic study

The synaptic interactions between terminals of allocorticostriatal and thalamostriatal fibers and the cholinergic neurons in the nucleus accumbens were investigated using degeneration and dual labelling immunocytochemistry in Wistar rats. The presumptive cholinergic neurons were labelled with antibodies directed against choline acetyltransferase and the afferent fibers were labelled anterogradely with Phaseolus vulgaris-leucoagglutinin. Fibers from the subiculum of the hippocampal formation and from the midline and intralaminar thalamus project densely into the medial nucleus accumbens where they overlap a relatively dense population of choline acetyltransferase-immunoreactive neurons. Varicosities containing Phaseolus vulgaris-leucoagglutinin juxtapose the immunoreactive neurons. To study the possibility that the cholinergic neurons could be the synaptic targets of these incoming fibers, the subiculum, the fornix, and the midline/intralaminar thalamus were lesioned in separate animals and brain sections were immunoprocessed for choline acetyltransferase and studied with the electron microscope. In addition, dual-labelling electron microscopic immunocytochemistry was employed. In total, 164 synaptic terminals from the subiculum/hippocampus and 130 from the midline/intralaminar thalamus were examined; all formed asymmetrical synaptic specializations. No hippocampal endings were seen to contact the somata or primary dendrites of the choline acetyltransferase-immunoreactive neurons; however, three were found in synaptic contact with distal, immunolabelled dendritic shafts. Most hippocampal terminals established contacts with unlabelled spines. Fifteen percent of the thalamic endings were found to synapse on the somata and the primary and distal dendrites of the choline acetyltransferase-immunoreactive neurons. The remaining thalamic terminals established synaptic junctions with small unlabelled dendrites or spines. These findings have important implications not only for our understanding of the synaptic organization of the hippocampal and thalamic projections to the nucleus accumbens, but also for the contribution of the cholinergic neurons to the circuitry of this nucleus.

Spiny neurons lacking choline acetyltransferase immunoreactivity are major targets of cholinergic and catecholaminergic terminals in rat striatum

The ultrastructural substrate for functional interactions between intrinsic cholinergic neurons and catecholaminergic afferents to the caudate-putamen nucleus and nucleus accumbens septi (NAS) was investigated immunocytochemically. Single sections of glutaraldehyde-fixed rat brain were processed 1) for the immunoperoxidase labeling of a rat monoclonal antibody against the acetylcholine-synthesizing enzyme choline acetyltransferase (CAT) and 2) for the immunoautoradiographic localization of a rabbit polyclonal antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). The ultrastructural morphology and cellular associations did not significantly differ in the caudate-putamen versus NAS. Immunoperoxidase reaction for CAT versus NAS. Immunoperoxidase reaction for CAT was seen in perikarya, dendrites, and terminals, whereas immunoautoradiography for TH was in terminals. The perikarya and dendrites immunolabeled for CAT were large, sparsely spiny, and postsynaptic mainly to unlabeled axon terminals. Only 2-3% of the CAT-labeled terminals (n = 136) and less than 1% of the TH-labeled terminals (n = 86) were apposed to, or formed synapses with, perikarya or dendrites immunoreactive for CAT. Most unlabeled and all labeled terminals formed symmetric synapses. In the same sample, 18% of the CAT and 16% of the TH-labeled terminals were directly apposed to each other. Unlabeled dendritic shafts received the major (40% for CAT versus 23% for TH) synaptic input from cholinergic terminals, while unlabeled spines received the major (47% for TH versus 23% for CAT) synaptic input from catecholaminergic terminals. Neither the unlabeled dendrites or spines received detectable convergent input from CAT and TH-labeled terminals. Thirteen percent of the CAT-labeled and 14% of TH-labeled terminals were in apposition to unlabeled terminals forming asymmetric, presumably excitatory, synapses with unlabeled dendritic spines. We conclude that in both the caudate-putamen and NAS cholinergic and catecholaminergic terminals 1) form symmetric, most likely inhibitory, synapses primarily with non-cholinergic neurons, 2) differentially synapse on shafts or spines of separate dendrites, and 3) have axonal appositions suggesting the possibility of presynaptic physiological interactions. These results support the hypothesis that the cholinergic-dopaminergic balance in striatal function may be mediated through inhibition of separate sets of spiny projection neurons with opposing excitatory and inhibitory functions.