Confocal laser scanning microscopy and ultrastructural study of VGLUT2 thalamic input to striatal projection neurons in rats

We examined thalamic input to striatum in rats using immunolabeling for the vesicular glutamate transporter (VGLUT2). Double immunofluorescence viewed with confocal laser scanning microscopy (CLSM) revealed that VGLUT2+ terminals are distinct from VGLUT1+ terminals. CLSM of Phaseolus vulgaris‐leucoagglutinin (PHAL)‐labeled cortical or thalamic terminals revealed that VGLUT2 is rare in corticostriatal terminals but nearly always present in thalamostriatal terminals. Electron microscopy revealed that VGLUT2+ terminals made up 39.4% of excitatory terminals in striatum (with VGLUT1+ corticostriatal terminals constituting the rest), and 66.8% of VGLUT2+ terminals synapsed on spines and the remainder on dendrites. VGLUT2+ axospinous terminals had a mean diameter of 0.624 μm, while VGLUT2+ axodendritic terminals a mean diameter of 0.698 μm. In tissue in which we simultaneously immunolabeled thalamostriatal terminals for VGLUT2 and striatal neurons for D1 (with about half of spines immunolabeled for D1), 54.6% of VGLUT2+ terminals targeted D1+ spines (i.e., direct pathway striatal neurons), and 37.3% of D1+ spines received VGLUT2+ synaptic contacts. By contrast, 45.4% of VGLUT2+ terminals targeted D1‐negative spines (i.e., indirect pathway striatal neurons), and only 25.8% of D1‐negative spines received VGLUT2+ synaptic contacts. Similarly, among VGLUT2+ axodendritic synaptic terminals, 59.1% contacted D1+ dendrites, and 40.9% contacted D1‐negative dendrites. VGLUT2+ terminals on D1+ spines and dendrites tended to be slightly smaller than those on D1‐negative spines and dendrites. Thus, thalamostriatal terminals contact both direct and indirect pathway striatal neurons, with a slight preference for direct. These results are consistent with physiological studies indicating slightly different effects of thalamic input on the two types of striatal projection neurons. J. Comp. Neurol., 521:1354–1377, 2013. © 2012 Wiley Periodicals, Inc.     

Ultrastructural study of nitric oxide synthase-containing striatal neurons and their relationship with parvalbumin-containing neurons in rats

Single- and double-label electron microscopic immunocytochemistry was used to examine the ultrastructure of striatal neurons containing nitric oxide synthase (NOS⁺) and evaluate the synaptic relationship of NOS⁺ striatal neurons with those containing parvalbumin (PV⁺). In both the single-label and double-label studies, NOS⁺ perikarya were observed to possess polylobulated nuclei. In the single-label studies, NOS⁺ terminals were seen forming synaptic contacts with dendritic shafts and dendritic spines that did not contain NOS, but not with NOS⁺ perikarya or dendrites. In the double-label studies (using diaminobenzidine and silver intensified immunogold as markers), nitric oxide synthase and parvalbumin immunoreactions were found in two different populations of medium-sized aspiny striatal neurons. The PV⁺ axon terminals were seen forming symmetric synapses on the dendritic spines of neurons devoid of PV or NOS labeling, on PV⁺ dendrites, and on NOS⁺ soma and dendrites. In contrast, NOS⁺ terminals were not observed to form synaptic contacts with the dendrites or soma of either PV⁺ or NOS⁺ neurons. These findings suggest that NOS⁺ striatal interneurons form synaptic contact with the spines and presumably the dendrites of striatal projection neurons, but not with the dendrites or soma of PV⁺ or NOS⁺ striatal interneurons. NOS⁺ neurons do, however, receive synaptic input from PV⁺ neurons.     

Localization of the M2 muscarinic cholinergic receptor in dendrites,cholinergic terminals,and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis

Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. The present investigation used immunocytochemistry at the electron microscopic level to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine whether M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R immunoreactivity (M2R‐ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R–negative. About 95% of dendritic shafts and 60% of dendritic spines were M2 immunoreactive (M2R⁺). Some M2R⁺ dendrites had spines, suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R‐ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R⁺ terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R⁺. The main targets of M2R⁺ terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R⁺ dendritic shafts. M2R‐ir was also seen in VAChT⁺ cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R‐mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites as well as regulating release of glutamate, γ‐aminobutyric acid, and acetylcholine from presynaptic axon terminals. J. Comp. Neurol. 524:2400–2417, 2016. © 2016 Wiley Periodicals, Inc.     

Reduced striatal acetylcholine efflux in the R6/2 mouse model of Huntington's disease: an examination of the role of altered inhibitory and excitatory mechanisms

Huntington's disease (HD) is a genetic neurodegenerative disorder that is characterized by the progressive onset of cognitive, psychiatric, and motor symptoms. In parallel, the neuropathology of HD is characterized by progressive loss of projection neurons in cortex and striatum; striatal cholinergic interneurons are relatively spared. Nonetheless, there is evidence that striatal acetylcholine (ACh) function is altered in HD. The present study is the first to examine striatal ACh function in awake, behaving animals, using the R6/2 mouse model of HD, which is transgenic for exon 1 of the mutant huntingtin gene. Physiological levels of extracellular striatal ACh were monitored in R6/2 mice and wild type controls using in vivo microdialysis. Results indicate that spontaneous ACh release is reduced in R6/2 mice relative to controls. Intrastriatal application of the GABA_A antagonist bicuculline methiodide (10.0 μM) significantly elevated ACh levels in both R6/2 mice and wild type controls, while overall ACh levels were reduced in the R6/2 mice compared to the wild type group. In contrast, systemic administration of the D₁ dopamine receptor partial agonist, SKF-38393 (10.0 mg/kg, IP), elevated ACh levels in control animals, but not R6/2 mice. Taken together, the present results suggest that GABA-mediated inhibition of striatal ACh release is intact in R6/2 mice, further demonstrating that cholinergic interneurons are capable of increased ACh release, whereas D₁ receptor-dependent activation of excitatory inputs to striatal cholinergic interneurons is dysfunctional in R6/2 mice. Reduced levels of extracellular striatal ACh in HD may reflect abnormalities in the excitatory innervation of cholinergic interneurons, which may have implications ACh-dependent processes that are altered in HD, including corticostriatal plasticity.     

Characterization of the excitatory amino acid receptor-mediated release of [H]acetylcholine from rat striatal slices

The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [³H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [³H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-dl-aspartate = ibotenate >l-glutamate >l-aspartate cysteate > kainate = quisqualate.The excitatory amino acid receptor mediating [³H]acetylcholine release resembles the N-methyl-d-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate.The release of [³H]acetylcholine evoked by N-methyl-dl-aspartate was blocked by tetrodotoxin (0.5 μM). Moreover, N-methyl-dl-aspartate failed to evoke [³H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.     

Dopamine-acetylcholine interaction in the rat striatum: A dual-labeling immunocytochemical study

The relationship between cholinergic neurons and dopaminergic axons in the rat striatum was examined by a dual-labeling immunocytochemical method. Cholinergic neurons were identified by their immunoreactivity for choline acetyltransferase (ChAT), and dopaminergic axon terminals were identified by their positive immunoreactivity for tyrosine hydroxylase (TH). Electron microscopic analysis of dual-labeled sections revealed that while most TH-positive terminals formed synapses with unlabeled striatal neurons and dendrites, a number of TH-positive terminals formed close appositions, highly suggestive of synapses, with both large and small dendrites as well as somata of ChAT-positive neurons. Tight appositions were also found between TH-positive terminals and ChAT-positive terminals. Moreover, TH-positive terminals and ChAT-positive terminals were found to form synapses with common dendrites of unlabeled striatal neurons. These results indicated that 1) dopaminergic axon terminals could interact directly with striatal cholinergic interneurons via tight appositions with distances comparable to conventional synapses; and 2) there is a convergence of dopaminergic and cholinergic axon terminals on noncholinergic striatal neurons.     

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models

Cannabinoid receptor 1 (CB₁ receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB₁ expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB₁ signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB₁ downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi‐quantitative immunohistochemistry, we confirmed previous studies showing that CB₁ expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB₁ is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)‐expressing interneurons while remaining unchanged in parvalbumin‐ and calretinin‐expressing interneurons. CB₁ downregulation in striatal NPY/nNOS‐expressing interneurons occurs in R6/2 mice, Hdh^Q150/Q150 mice and the caudate nucleus of patients with HD. In R6/2 mice, CB₁ downregulation in NPY/nNOS‐expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro‐survival signaling molecule cAMP response element‐binding protein in NPY/nNOS‐expressing interneurons. Loss of CB₁ signaling in NPY/nNOS‐expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.     

Expression of the mRNAs encoding for the vesicular glutamate transporters 1 and 2 in the rat thalamus

Vesicular glutamate transporters (VGLUTs) are responsible for glutamate trafficking and for the subsequent regulated release of this excitatory neurotransmitter at the synapse. Three isoforms of the VGLUT have been identified, now known as VGLUT1, VGLUT2, and VGLUT3. Both VGLUT1 and VGLUT2 have been considered definitive markers of glutamatergic neurons, whereas VGLUT3 is expressed in nonglutamatergic neurons such as cholinergic striatal interneurons. It is widely believed that VGLUT1 and VGLUT2 are expressed in a complementary manner at the cortical and thalamic levels, suggesting that these glutamatergic neurons fulfill different physiological functions. In the present work, we analyzed the pattern of VGLUT1 and VGLUT2 mRNA expression at the thalamic level by using single and dual in situ hybridization. In accordance with current beliefs, we found significant expression of VGLUT2 mRNA in all the thalamic nuclei, while moderate expression of VGLUT1 mRNA was consistently found in both the principal relay and the association thalamic nuclei. Interestingly, individual neurons within these nuclei coexpressed both VGLUT1 and VGLUT2 mRNAs, suggesting that these individual thalamic neurons may have different ways of trafficking glutamate. These results call for a reappraisal of the previously held concept regarding the mutually exclusive distribution of VGLUT transporters in the central nervous system.     

Parvalbumin‐producing striatal interneurons receive excitatory inputs onto proximal dendrites from the motor thalamus in male mice

In rodents, the dorsolateral striatum regulates voluntary movement by integrating excitatory inputs from the motor‐related cerebral cortex and thalamus to produce contingent inhibitory output to other basal ganglia nuclei. Striatal parvalbumin (PV)‐producing interneurons receiving this excitatory input then inhibit medium spiny neurons (MSNs) and modify their outputs. To understand basal ganglia function in motor control, it is important to reveal the precise synaptic organization of motor‐related cortical and thalamic inputs to striatal PV interneurons. To examine which domains of the PV neurons receive these excitatory inputs, we used male bacterial artificial chromosome transgenic mice expressing somatodendritic membrane–targeted green fluorescent protein in PV neurons. An anterograde tracing study with the adeno‐associated virus vector combined with immunodetection of pre‐ and postsynaptic markers visualized the distribution of the excitatory appositions on PV dendrites. Statistical analysis revealed that the density of thalamostriatal appositions along the dendrites was significantly higher on the proximal than distal dendrites. In contrast, there was no positional preference in the density of appositions from axons of the dorsofrontal cortex. Population observations of thalamostriatal and corticostriatal appositions by immunohistochemistry for pathway‐specific vesicular glutamate transporters confirmed that thalamic inputs preferentially, and cortical ones less preferentially, made apposition on proximal dendrites of PV neurons. This axodendritic organization suggests that PV neurons produce fast and reliable inhibition of MSNs in response to thalamic inputs and process excitatory inputs from motor cortices locally and plastically, possibly together with other GABAergic and dopaminergic dendritic inputs, to modulate MSN inhibition.     

Synaptic microcircuitry of tyrosine hydroxylase-containing neurons and terminals in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys

A population of tyrosine hydroxylase (TH)-containing neurons that is up-regulated after lesion of the nigrostriatal dopaminergic pathway has been described in the primate striatum. The goal of this study was to examine the morphology, synaptology, and chemical phenotype of these neurons and TH-immunoreactive (-ir) terminals in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys. TH-ir perikarya were small (10-12 microm), displayed nuclear invaginations, and received very few synaptic inputs. On the other hand, TH-containing dendrites were typically large in diameter (>1.0 microm) and received scarce synaptic innervation from putative excitatory and inhibitory terminals forming asymmetric and symmetric synapses, respectively. More than 70% of TH-positive intrastriatal cell bodies were found in the caudate nucleus and the precommissural putamen, considered as the associative functional territories of the primate striatum. Under 10% of these cells displayed calretinin immunoreactivity. TH-ir terminals rarely formed clear synaptic contacts, except for a few that established asymmetric axodendritic synapses. Almost two-thirds of TH-containing boutons displayed gamma-aminobutyric acid (GABA) immunoreactivity in the striatum of parkinsonian monkeys, whereas under 5% did so in the normal striatum. These findings provide strong support for the existence of a population of putative catecholaminergic interneurons in the associative territory of the striatum in parkinsonian monkeys. Their sparse synaptic innervation raises interesting issues regarding synaptic and nonsynaptic mechanisms involved in the regulation and integration of these neurons in the striatal microcircuitry. Finally, the coexpression of GABA in TH-positive terminals in the striatum of dopamine-depleted monkeys suggests dramatic neurochemical changes in the catecholaminergic modulation of striatal activity in Parkinson's disease.     

Transient global ischemia in rats yields striatal projection neuron and interneuron loss resembling that in Huntington's disease

The various types of striatal projection neurons and interneurons show a differential pattern of loss in Huntington's disease (HD). Since striatal injury has been suggested to involve similar mechanisms in transient global brain ischemia and HD, we examined the possibility that the patterns of survival for striatal neurons after transient global ischemic damage to the striatum in rats resemble that in HD. The perikarya of specific types of striatal interneurons were identified by histochemical or immunohistochemical labeling while projection neuron abundance was assessed by cresyl violet staining. Projectionneuron survival was assessed by neurotransmitter immunolabeling of their efferent fibers in striatal target areas. The relative survival of neuron types was determined quantitatively within the region of ischemic damage, and the degree of fiber loss in striatal target areas was quantified by computer-assisted image analysis. We found that NADPHd(+) and cholinergic interneurons were largely unaffected, even in the striatal area of maximal damage. Parvalbumin interneurons, however, were as vulnerable as projection neurons. Among immunolabeled striatal projection systems, striatoentopeduncular fibers survived global ischemia better than did striatopallidal or striatonigral fibers. The order of vulnerability observed in this study among the striatal projection systems, and the resistance to damage shown by NADPHd(+) and cholinergic interneurons, is similar to that reported in HD. The high vulnerability of projection neurons and parvalbumin interneurons to global ischemia also resembles that seen in HD. Our results thus indicate that global ischemic damage to striatum in rat closely mimics HD in its neuronal selectivity, which supports the notion that the mechanisms of injury may be similar in both.     

Plastic and behavioral abnormalities in experimental Huntington's disease: a crucial role for cholinergic interneurons

Huntington's disease (HD) is a fatal hereditary neurodegenerative disease causing degeneration of striatal spiny neurons, whereas cholinergic interneurons are spared. This cell-type specific pathology produces an array of abnormalities including involuntary movements, cognitive impairments, and psychiatric disorders. Although the genetic mutation responsible for HD has been identified, little is known about the early synaptic changes occurring within the striatal circuitry at the onset of clinical symptoms. We therefore studied the synaptic plasticity of spiny neurons and cholinergic interneurons in two animal models of early HD. As a pathogenetic model, we used the chronic subcutaneous infusion of the mitochondrial toxin 3-nitropropionic acid (3-NP) in rats. This treatment caused striatal damage and impaired response flexibility in the cross-maze task as well as defective extinction of conditioned fear suggesting a perseverative behavior. In these animals, we observed a loss of depotentiation in striatal spiny neurons and a lack of long-term potentiation (LTP) in cholinergic interneurons. These abnormalities of striatal synaptic plasticity were also observed in R6/2 transgenic mice, a genetic model of HD, indicating that both genetic and phenotypic models of HD show cell-type specific alterations of LTP. We also found that in control rats, as well as in wild-type (WT) mice, depotentiation of spiny neurons was blocked by either scopolamine or hemicholinium, indicating that reversal of LTP requires activation of muscarinic receptors by endogenous acetylcholine. Our findings suggest that the defective plasticity of cholinergic interneurons could be the primary event mediating abnormal functioning of striatal circuits, and the loss of behavioral flexibility typical of early HD might largely depend on cell-type specific plastic abnormalities.     

Immunogold characteristics of VGLUT3‐positive GABAergic nerve terminals suggest corelease of glutamate

There is compelling evidence that glutamate can act as a cotransmitter in the mammalian brain. Interestingly, the third vesicular glutamate transporter (VGLUT3) is primarily found in neurons that were anticipated to be nonglutamatergic. Whereas the function of VGLUT3 in acetylcholinergic and serotoninergic neurons has been elucidated, the role of VGLUT3 in neurons releasing gamma‐aminobutyric acid (GABA) is not settled. We have previously shown that VGLUT3 is found together with the vesicular GABA transporter (VIAAT) on synaptic vesicle membranes in the hippocampus. Now we provide novel electron microscopic data from the rat hippocampus suggesting that glutamate is enriched in inhibitory nerve terminals containing VGLUT3 compared to those lacking VGLUT3. The opposite was found for GABA; VGLUT3‐positive inhibitory terminals contained lower density of GABA labeling compared to VGLUT3‐negative inhibitory terminals. In addition, semiquantitative confocal immunofluorescence showed that N‐methyl‐D‐aspartate (NMDA)‐receptor labeling was present more frequently in VGLUT3‐positive/VIAAT‐positive synapses versus in VGLUT3‐negative/VIAAT‐positive synapses. Electron microscopic immunogold data further suggest that NMDA receptors are enriched in VGLUT3 containing inhibitory terminals. Our data reveal new chemical characteristics of a subset of GABAergic interneurons in the hippocampus. The analyses suggest that glutamate is coreleased with GABA from hippocampal basket cell‐synapses to act on NMDA receptors. J. Comp. Neurol. 523:2698–2713, 2015. © 2015 Wiley Periodicals, Inc.     

Extracerebellar role for Cerebellin1: Modulation of dendritic spine density and synapses in striatal medium spiny neurons

Cerebellin1 (Cbln1) is a secreted glycoprotein that was originally isolated from the cerebellum and subsequently found to regulate synaptic development and stability. Cbln1 has a heterogeneous distribution in brain, but the only site in which it has been shown to have central effects is the cerebellar cortex, where loss of Cbln1 causes a reduction in granule cell‐Purkinje cell synapses. Neurons of the thalamic parafascicular nucleus (PF), which provide glutamatergic projections to the striatum, also express high levels of Cbln1. We first examined Cbln1 in thalamostriatal neurons and then determined if cbln1 knockout mice exhibit structural deficits in striatal neurons. Virtually all PF neurons express Cbln1‐immunoreactivity (‐ir). In contrast, only rare Cbln1‐ir neurons are present in the central medial complex, the other thalamic region that projects heavily to the dorsal striatum. In the striatum Cbln1‐ir processes are apposed to medium spiny neuron (MSN) dendrites; ultrastructural studies revealed that Cbln1‐ir axon terminals form axodendritic synapses with MSNs. Tract‐tracing studies found that all PF cells retrogradely labeled from the striatum express Cbln1‐ir. We then examined the dendritic structure of Golgi‐impregnated MSNs in adult cbln1 knockout mice. MSN dendritic spine density was markedly increased in cbln1^−/− mice relative to wildtype littermates, but total dendritic length was unchanged. Ultrastructural examination revealed an increase in the density of MSN axospinous synapses in cbln1^−/− mice, with no change in postsynaptic density length. Thus, Cbln1 determines the dendritic structure of striatal MSNs, with effects distinct from those seen in the cerebellum. J. Comp. Neurol. 518:2525–2537, 2010. © 2010 Wiley‐Liss, Inc.     

Cellular localization and development of neuronal intranuclear inclusions in striatal and cortical neurons in R6/2 transgenic mice

The cellular localization and development of neuronal intranuclear inclusions (NIIs) in cortex and striatum of R6/2 HD transgenic mice were studied to ascertain the relationship of NIIs to symptom formation in these mice and gain clues regarding the possible relationship of NII formation to neuropathology in Huntington's disease (HD). All NIIs observed in R6/2 mice were ubiquitinated, and no evidence was observed for a contribution to them from wild-type huntingtin; they were first observed in cortex and striatum at 3.5 weeks of age. In cortex, NIIs increased rapidly in size and prevalence after their appearance. Generally, cortical projection neurons developed NIIs more rapidly than cortical interneurons containing calbindin or parvalbumin. Few cortical somatostatinergic interneurons, however, formed NIIs. In striatum, calbindinergic projection neurons and parvalbuminergic interneurons rapidly formed NIIs, but they formed more gradually in cholinergic interneurons, and few somatostatinergic interneurons developed NIIs. Striatal NIIs tended to be smaller than those in cortex. The early accumulation of NIIs in cortex and striatum in R6/2 mice is consistent with the early appearance of motor and learning abnormalities in these mice, and the eventual pervasiveness of NIIs at ages at which severe abnormalities are evident is consistent with their contribution to a neuronal dysfunction underlying the abnormalities. That cortex develops larger NIIs than striatum, however, is inconsistent with the preferential loss of striatal neurons in HD but is consistent with recent evidence of early morphological abnormalities in cortical neurons in HD. That calbindinergic and parvalbuminergic striatal neurons develop large NIIs is consistent with a contribution of nuclear aggregate formation to their high degree of vulnerability in HD.     

Special function of nestin~+ neurons in the medial septum-diagonal band of Broca in adult rats

Nestin+ neurons have been shown to express choline acetyltransferase(ChAT) in the medial septum-diagonal band of Broca in adult rats. This study explored the projection of nestin+ neurons to the olfactory bulb and the time course of nestin+ neurons in the medial septum-diagonal band of Broca in adult rats during injury recovery after olfactory nerve transection. This study observed that all nestin+ neurons were double-labeled with ChAT in the medial septum-diagonal band of Broca. Approximately 53.6% of nestin+ neurons were projected to the olfactory bulb and co-labeled with fast blue. A large number of nestin+ neurons were not present in each region of the medial septum-diagonal band of Broca. Nestin+ neurons in the medial septum and vertical limb of the diagonal band of Broca showed obvious compensatory function. The number of nestin+ neurons decreased to a minimum later than nestin–/ChAT+ neurons in the medial septum-diagonal band of Broca. The results suggest that nestin+ cholinergic neurons may have a closer connection to olfactory bulb neurons. Nestin+ cholinergic neurons may have a stronger tolerance to injury than Nestin–/ChAT+ neurons. The difference between nestin+ and nestin–/ ChAT+ neurons during the recovery process requires further investigations.     

Cortical inputs to m2-immunoreactive striatal interneurons in rat and monkey

Previous anatomical studies have been unsuccessful in demonstrating significant cortical inputs to cholinergic and somatostatinergic striatal interneurons in rats. On the other hand, electrophysiological studies have shown that cortical stimulation induces monosynaptic EPSPs in cholinergic interneurons. It has been proposed that the negative anatomical findings might have been the result of incomplete labeling of distal dendrites. In the present study, we reinvestigated this issue using m2 muscarinic receptor antibodies as a selective marker for cholinergic and somatostatinergic interneurons in the striatum. This was combined with injections of either the anterograde tracer biotinylated dextran amine (BDA) in the monkey prefrontal cortex or aspiration lesion of the sensorimotor cortex in rats. The results showed that, in both species, a small percentage (1-2%) of cortical terminals make asymmetric synaptic contacts with m2-immunoreactive interneurons in the striatum. Interestingly, the majority of these synapses are onto small dendritic spines or spine-like appendages, as opposed to dendritic shafts and/or cell bodies. Thus, m2-containing striatal interneurons do receive direct cortical inputs and can, therefore, integrate and modulate cortical information flow through the striatum. Although the density of cortical terminals in contact with individual striatal interneurons is likely to be relatively low compared to the massive cortical input to projection neurons, both cholinergic and somatostatinergic interneurons display intrinsic properties that allow even small and distal inputs to influence their overall state of neuronal activity.     

Cholinergic modulation of striatal nitric oxide‐producing interneurons

Striatal GABAergic interneurons that express nitric oxide synthase—so‐called low‐threshold spike interneurons (LTSIs)—play several key roles in the striatum. But what drives the activity of these interneurons is less well defined. To fill this gap, a combination of monosynaptic rabies virus mapping (msRVm), electrophysiological and optogenetic approaches were used in transgenic mice in which LTSIs expressed either Cre recombinase or a fluorescent reporter. The rabies virus studies revealed a striking similarity in the afferent connectomes of LTSIs and neighboring cholinergic interneurons, particularly regarding connections arising from the parafascicular nucleus of the thalamus and cingulate cortex. While optogenetic stimulation of cingulate inputs excited both cholinergic interneurons and LTSIs, thalamic stimulation excited cholinergic interneurons, but inhibited LTSIs. This inhibition was dependent on cholinergic interneurons and had two components: a previously described GABAergic element and one that was mediated by M4 muscarinic acetylcholine receptors. In addition to this phasic signal, cholinergic interneurons tonically excited LTSIs through a distinct, M1 muscarinic acetylcholine receptor pathway. This coordinated cholinergic modulation of LTSIs predisposed them to rhythmically burst in response to phasic thalamic activity, potentially reconfiguring striatal circuitry in response to salient environmental stimuli.