François Laplante

Distinct electrophysiological properties of glutamatergic, cholinergic and GABAergic rat septohippocampal neurons: novel implications for hippocampal rhythmicity

The medial septum‐diagonal band complex (MSDB) contains cholinergic and non‐cholinergic neurons known to play key roles in learning and memory processing, and in the generation of hippocampal theta rhythm. Electrophysiologically, several classes of neurons have been described in the MSDB, but their chemical identity remains to be fully established. By combining electrophysiology with single‐cell RT‐PCR, we have identified four classes of neurons in the MSDB in vitro. The first class displayed slow‐firing and little or no Ih, and expressed choline acetyl‐transferase mRNA (ChAT). The second class was fast‐firing, had a substantial Ih and expressed glutamic acid decarboxylase 67 mRNA (GAD67), sometimes co‐localized with ChAT mRNAs. A third class exhibited fast‐ and burst‐firing, had an important Ih and expressed GAD67 mRNA also occasionally co‐localized with ChAT mRNAs. The ionic mechanism underlying the bursts involved a low‐threshold spike and a prominent Ih current, conductances often associated with pacemaker activity. Interestingly, we identified a fourth class that expressed transcripts solely for one or two of the vesicular glutamate transporters (VGLUT1 and VGLUT2), but not ChAT or GAD. Some putative glutamatergic neurons displayed electrophysiological properties similar to ChAT‐positive slow‐firing neurons such as the occurrence of a very small Ih, but nearly half of glutamatergic neurons exhibited cluster firing with intrinsically generated voltage‐dependent subthreshold membrane oscillations. Neurons belonging to each of the four described classes were found among septohippocampal neurons by retrograde labelling. We provide results suggesting that slow‐firing cholinergic, fast‐firing and burst‐firing GABAergic, and cluster‐firing glutamatergic neurons, may each uniquely contribute to hippocampal rhythmicity in vivo.

ACETYLCHOLINE RELEASE IS ELICITED IN THE VISUAL CORTEX, BUT NOT IN THE PREFRONTAL CORTEX, BY PATTERNED VISUAL STIMULATION: A DUAL IN VIVO MICRODIALYSIS STUDY WITH FUNCTIONAL CORRELATES IN THE RAT BRAIN

By its projections to the primary visual and the prefrontal cortices, the basal forebrain cholinergic system is involved in cognitive processing of sensory stimuli. It has been suggested that visual stimulus-induced cholinergic activation of the visual cortex may exert a permissive role on thalamocortical inputs. However, it is not known if visual stimulation elicits cholinergic activation of high-order brain areas in the absence of attentional need. In the present study, we measured the effects of patterned visual stimulation (horizontal grating) on the release of acetylcholine with dual-probe in vivo microdialysis in the visual and the prefrontal cortices of anesthetized rats. We also used retrograde tracing to determine the anatomical relationships of cholinergic neurons with neurons of the visual system and the prefrontal cortex. Finally, we evaluated a functional correlate of this stimulation, namely c-fos immunolabeling. Patterned visual stimulation elicited significant increases in acetylcholine release in the visual cortex, accompanied by an increased number of c-fos immunoreactive neurons in this brain area. In contrast, in the prefrontal cortex, neither the level of acetylcholine release nor the number of c-fos immunoreactive neurons was significantly changed because of the stimulation. Cholinergic basal forebrain neurons projecting to the visual or the prefrontal cortices were both localized within the horizontal limb of the diagonal band of Broca but were not immunoreactive for c-fos during visual stimulation. No parts of the visual system were found to directly project to these basal forebrain neurons. These results suggest the differential involvement of cholinergic projections in the integration of sensory stimuli, depending on the level of activity of the targeted cortical area.

Reduction in acetylcholine release in the hippocampus of dopamine D5 receptor-deficient mice

Activation of the dopamine D1-like receptor stimulates acetylcholine (ACh) release in the hippocampus, apparently through the molecularly defined d5 receptor. In the present study, we used a transgenic mouse completely deprived of functional d5 receptor (d5−/−) to confirm the role and elucidate the possible function of the d5 receptor subtype on hippocampal cholinergic neurotransmission. ACh release was measured using in vivo microdialysis in the mouse dorsal hippocampus of 4 months old homozygous (d5−/−), heterozygous (d5+/−), and the wild-type (d5+/+) littermates. Using the no net flux technique, a significant reduction in basal hippocampal ACh level was found in the d5−/− compared to d5+/− and d5+/+ mice. Moreover, the administration of SKF 38393, a D1-like receptor agonist, systemically (2.0 and 10.0 mg/kg ip), or locally through the dialysis probe (10 and 50 μM), produced a dose-dependent enhancement of ACh release in the d5+/+, a moderate stimulation in the d5+/− but had no effect in the d5−/− mice. Quantitative receptor autoradiography revealed significant increases in M1-like but not in M2-like muscarinic receptor binding sites in the hippocampal formation. These results confirm and extend the role of the d5 receptor in the modulation of hippocampal ACh release and provide evidence for long-term alteration of hippocampal cholinergic neurotransmission resulting from the absence of the d5 receptors including chronically reduced ACh release and change in M1-like receptor levels.

Effects of neonatal ventral hippocampal lesion in rats on stress-induced acetylcholine release in the prefrontal cortex

Excitotoxic neonatal ventral hippocampus (NVH) lesions in rats result in characteristic post‐pubertal hyper‐responsiveness to stress and cognitive abnormalities analogous to those described in schizophrenia and suggestive of alterations in dopamine (DA) neurotransmission. Converging lines of evidence also point to dysfunctions in the cortical cholinergic system in neuropsychiatric disorders. In previous studies, we observed alterations in dopaminergic modulation of acetylcholine (Ach) release in the prefrontal cortex (PFC) in post‐pubertal NVH‐lesioned rats. These two neurotransmitter systems are involved in the stress response as PFC release of DA and Ach is enhanced in response to some stressful stimuli. As adult NVH‐lesioned rats are behaviorally more reactive to stress, we investigated the effects of NVH lesions on tail‐pinch stress‐induced Ach and DA release in the PFC. Using in vivo microdialysis, we observed that tail‐pinch stress resulted in significantly greater increases in prefrontal cortical Ach release in post‐pubertal NVH‐lesioned rats (220% baseline) compared with sham‐operated controls (135% baseline). Systemic administration of the D1‐like receptor antagonist SCH 23390 (0.5 mg/kg i.p.) or the D2‐like receptor antagonist haloperidol (0.2 mg/kg i.p.), as well as intra‐PFC administration of the D2‐like antagonist sulpiride (100 µm), reduced stress‐induced Ach release in PFC of adult NVH‐lesioned rats. By contrast, intra‐PFC administration of SCH 23390 (100 µm) failed to affect stress‐induced Ach release in PFC of NVH‐lesioned rats. Interestingly, using in vivo voltammetry, stress‐induced stimulation of PFC DA release was found to be attenuated in adult NVH‐lesioned rats. Taken together, these data suggest developmentally specific reorganization of prefrontal cortical cholinergic innervation notably regarding its regulation by DA neurotransmission.

Alterations in dopaminergic modulation of prefrontal cortical acetylcholine release in post-pubertal rats with neonatal ventral hippocampal lesions

Excitotoxic lesion of the ventral hippocampus in neonatal rats is a putative animal model of schizophrenia with characteristic developmental abnormalities in dopaminergic neurotransmission and prefrontal cortical functions. Converging evidence also points to the involvement of the central cholinergic system in neuropsychiatric disorders. These two neurotransmitter systems are interlinked in the prefrontal cortex (PFC) where dopamine stimulates acetylcholine (ACh) release. In the present study, we investigated the role of dopamine in the developmental regulation of prefrontal cortical ACh release and the expression of nicotinic and muscarinic receptors in pre‐ and post‐pubertal rats with neonatal ibotenic acid‐induced lesions of the ventral hippocampus (NVH). In vivo microdialysis in the PFC revealed that systemic injections of the D1‐like receptor agonist (±)‐6‐chloro‐7,8‐dihydroxy‐1‐phenyl2,3,4,5‐tetrahydro‐1H‐3‐benzazepine hydrobromide (SKF 81297) (2.5 and 5.0 mg/kg i.p.) caused significantly higher ACh release in post‐pubertal NVH‐lesioned animals (250 and 300% baseline for 2.5 and 5.0 mg/kg, respectively) compared with post‐pubertal shams (150 and 220% baseline for 2.5 and 5.0 mg/kg, respectively). Most interestingly, while prefrontal cortical perfusion of SKF 81297 (100 and 250 µm) had no significant effect on ACh release in post‐pubertal sham‐operated animals, it significantly stimulated ACh release to approximately 250% baseline at both doses in post‐pubertal NVH‐lesioned animals. Receptor autoradiography demonstrated a significant and selective increase in M1‐like receptor binding sites in the infralimbic area of the PFC in the post‐pubertal NVH‐lesioned animals. For all experiments, significant differences between sham and NVH‐lesioned animals were observed only in post‐pubertal rats. These results suggest a developmentally specific reorganization of the prefrontal cortical cholinergic system involving D1‐like receptors in the NVH model.

Selective reduction in ventral hippocampal acetylcholine release in awake galanin-treated rats and galanin-overexpressing transgenic mice.

The neuropeptide galanin is an inhibitory modulator of hippocampal acetylcholine (ACh) release and cognitive functions. Anatomical evidence demonstrated some differences between the dorsal and ventral hippocampi notably in the expression of galanin receptor subtypes, and the neuronal population on which galanin-like immunoreactivity is expressed. This is suggestive of a differential role for this peptide in these two areas of the hippocampal formation. Using in vivo microdialysis, we investigated the role of galanin on ACh release in the dorsal and ventral hippocampi. Two models were studied: galanin-administered rats and transgenic mice over-expressing galanin (GAL-tg). In rats, galanin (2.0 and 10.0 microM) infused locally through the dialysis probe induced a significant decrease in ACh release in the ventral hippocampus, confirming previous findings, while no effect was seen in the dorsal hippocampus. Using the no net flux method, a significant reduction in ACh levels was noted only in the ventral hippocampus of GAL-tg compared to wild-type littermates. These results suggest that excess endogenous galanin can suppress basal ACh release, with anatomical specificity, to the ventral hippocampus. These results are of interest in the context of galanin receptor subtypes in the dorsal and ventral hippocampus, and the differential alterations of hippocampal subregions in neurological diseases such as Alzheimers dementia.

Cholinergic depletion in the nucleus accumbens: Effects on amphetamine response and sensorimotor gating

A delicate balance between dopaminergic and cholinergic activity in the ventral striatum or nucleus accumbens (N.Acc) appears to be important for optimal performance of a wide range of behaviours. While functional interactions between these systems are complex, some data suggest that acetylcholine in the N.Acc. may dampen the effects of excessive dopamine (DA) release. We proposed that a reduction in the density of cholinergic interneurons in the N.Acc would result in behavioural alterations suggestive of a hyper-responsiveness of the N.Acc DA system. The present study aimed to produce a sustainable depletion of cholinergic neurons in the N.Acc in the rat and study the effects of such lesions on DA-dependent behaviour. A novel saporin immunotoxin targeting choline acetyltransferase was microinjected bilaterally into the N.Acc of adult rats. We confirmed histologically that two weeks post-injection, animals show a local, selective depletion of cholinergic interneurons (mean cell loss of 44%). Cholinergic-depleted rats showed a marked increase in the locomotor activating effects of amphetamine. In addition, such lesions induced a disruption of sensorimotor gating processes, reflected in a reduction in the prepulse inhibition of the acoustic startle response, which was reversed by haloperidol. These data are suggestive of pronounced hyper-responsiveness of the meso-accumbens DA system which may be of relevance to the pathophysiology of schizophrenia, a condition where selective reduction in the number of ventral striatal cholinergic neurons has been demonstrated.

Alterations in Behavioral Responses to a Cholinergic Agonist in Post-Pubertal Rats with Neonatal Ventral Hippocampal Lesions: Relationship to Changes in Muscarinic Receptor Levels

Excitotoxic neonatal ventral hippocampal (NVH) lesion in rats is considered as a putative animal model of schizophrenia as lesioned animals show characteristic post-pubertal emergence of neurochemical and behavioral abnormalities analogous to some of those seen in this disease. Converging evidence points to the involvement of central cholinergic system in this neuropsychiatric disorder, and our previous studies have suggested that cholinergic neurotransmission may be altered in post-pubertal NVH lesioned rats. We investigated here muscarinic receptor reactivity in NVH lesioned animals by measuring the effects of the muscarinic receptor agonist oxotremorine on physiological responses known to be modulated by these receptors such as body temperature, salivation, tremor, pain, and prepulse inhibition of the acoustic startle (PPI). Quantitative receptor autoradiography revealed that post-pubertal NVH lesioned animals display increased levels of [3H]pirenzepine/M1-like and [3H]AFDX-384/M2-like receptor binding sites in the striatum, nucleus accumbens, and in subareas of the dorsal hippocampus. Moreover, in response to the systemic administration of oxotremorine (0.25 mg/kg), post-pubertal NVH lesioned rats exhibited increases in salivation and tremor, and a greater reduction in body temperature compared to sham control animals. Increases in the hot-plate latency were also observed suggesting enhanced antinociceptive effects of oxotremorine in post-pubertal NVH lesioned animals. Finally, oxotremorine (0.1 and 0.25 mg/kg) disrupted PPI in post-pubertal sham control rats while the muscarinic receptor antagonist biperiden (0.5 and 1.0 mg/kg) normalized this behavior in NVH lesioned rats. Taken together, these findings reveal that post-pubertal NVH lesioned rats display enhanced muscarinic receptor responsiveness, which may relate to some behavioral abnormalities reported in this animal model.

Cholinergic depletion in nucleus accumbens impairs mesocortical dopamine activation and cognitive function in rats

In rats, selective depletion of the cholinergic interneurons in the ventral striatum (nucleus accumbens or N.Acc.) results in heightened behavioural sensitivity to amphetamine and impaired sensorimotor gating processes, suggesting a hyper-responsiveness to dopamine (DA) activity in the N.Acc. We hypothesized that local cholinergic depletion may also trigger distal functional alterations, particularly in prefrontal cortex (PFC). Adult male Sprague-Dawley rats were injected bilaterally in the N.Acc. with an immunotoxin targeting choline acetyltransferase. Two weeks later, cognitive function was assessed using the delayed alternation paradigm in the T-maze. The rats were then implanted with voltammetric recording electrodes in the ventromedial PFC to measure in vivo extracellular DA release in response to mild tail pinch stress. The PFC was also examined for density of tyrosine hydroxylase (TH)-labelled varicosities. In another cohort of control and lesioned rats, we measured post mortem tissue content of DA. Depletion of cholinergic neurons (restricted to N.Acc.) significantly impaired delayed alternation performance across delay intervals. While (basal) post mortem indices of PFC DA function were unaffected by N.Acc. lesions, in vivo mesocortical DA activation was markedly reduced; this deficit correlated significantly with cognitive impairments. TH-labelled varicosities however, were unaffected in cortical layer V relative to controls. These data suggest that selective depletion of cholinergic interneurons in N.Acc. triggers widespread functional impairments in mesocorticolimbic DA function and cognition. The possible relevance of these findings is also discussed in relation to schizophrenia, where reduced density of cholinergic neurons in ventral striatum has been reported.

Sex differences in the effects of perinatal anoxia on dopamine function in rats

Birth complications involving reduced oxygen to the fetus pose risks for neurodevelopmental disorders like schizophrenia and ADHD, which involve central dopamine (DA) dysfunction and also show gender differences in incidence or severity. Here, we examine possible sex differences in the long-term consequences of perinatal anoxia in the rat, on central DA systems and DA-mediated behaviour. As adults, sensorimotor gating (prepulse inhibition, PPI) was differentially affected by anoxia in males and females, tending to be impaired only in males. Apomorphine-induced suppression of PPI was especially pronounced in males. Anoxia caused increases in amygdala DA levels in both sexes. However, sex-specific changes in DA and metabolite levels in prefrontal cortex and nucleus accumbens were found, suggesting a possible basis for some of the observed gender biases in certain neurodevelopmental disorders, sensitive to birth hypoxia.