Hypoglycemic brain injury is a common and serious complication of insulin therapy in diabetic individuals 1 2 . Studies suggest that acute or chronic hypoglycemia leads to neurological dysfunction and injury. Severe hypoglycemia triggers a cascade of events in vulnerable neurons that culminate in cell death even after glucose normalization 3 4 5 . Children and adults exposed to hypoglycemia can develop long-term impairment of cognitive function 6 and are at risk of epilepsy.

Altered neurotransmitter action appears to play a role in hypoglycemic brain dysfunction 7 8 9 . Muscarinic acetylcholine receptors play important roles in many fundamental central functions including higher cognitive processes and modulation of extrapyramidal motor activity. Synaptic ACh levels are known to be regulated by the activity of presynaptic muscarinic autoreceptors mediating inhibition of ACh release. In terms of the contribution of cholinergic cerebellar abnormalities to mental function, early reports of cerebellar abnormalities in autism 10 and of intellectual and behavioural abnormalities in patients with cerebellar damage 11 originally suggested a cognitive role for the cerebellum. Since then, many studies have confirmed that the cerebellum contributes to cognitive and other non-motor functions. There is thus increasing evidence that the cerebellum is involved in cognition, behaviour and emotion 12 . Cerebellar dysfuncton is associated with poor fine motor skills, hypotonia 13 . Alterations in glucose utilization are known to occur in the important regions of brain connected with learning and memory 14 15 .

Receptors activate a multitude of signaling pathways important for modulating neuronal excitability, synaptic plasticity and feedback regulation of ACh release 16 . In the cerebellum, nicotinic acetylcholine receptors mediate the release of glutamate 17 , GABA 18 19 and norepinephrine 20 . Thus, these receptors significantly influence the activity within the cerebellar circuitry, and any deregulation of this activity contributes to functional disorders involving the cerebellum.

The altered levels of neurotransmitter in specific brain areas in patients with diabetes mellitus 21 and in animals with experimental diabetes 22 23 24 25 26 27 have been documented and implicated in the CNS disorders. Recently we have reported that muscarinic M1 receptor gene expressions were decreased in the cerebral cortex, brainstem, hypothalamus and pancreatic islets of STZ induced diabetic rats and insulin modulates the binding parameters and gene expression 28 29 .

Moderate hypoglycemia is known to have significant impact on functions of the central nervous system, and any differential effect of hypoglycemia on the peripheral nervous system may offer insights into the metabolic requirements of central and peripheral neurons 30 . In a case of episodic bilateral cerebellar dysfunction caused by hypoglycemia, quantitative dynamic PET study demonstrated decreased glucose uptake-to-utilization ratio and increased leak of glucose in the cerebellum indicating that cerebellum is not invariably resistant to hypoglycemia 31 . Studies from our laboratory have demonstrated that cerebellum is susceptible to hypoglycemia 32 33 . Studies on damages of the central nervous system under conditions of hypoglycemia are very important for clinical medicine. The main objective of the present study was to determine whether hypoglycemia as a consequence of insulin therapy in diabetes altered the binding parameters of Muscarinic M1, M3 receptors and gene expression of α7nAchR, AchE, ChAT and GLUT3 in the cerebellum and AchE activity in the muscle of experimental rats.

Male adult Wistar rats of 200-250 g body weight were used for all experiments. Animals were divided into the following groups as (i) control (C), (ii) diabetic (D), (iii) insulin-induced hypoglycemia in diabetic rats (diabetic + IIH) and (iv) insulin-induced hypoglycemia in control rats (control + IIH). Each group consisted of 6-8 rats. They were housed in separate cages under 12-h light and 12-h dark periods and were maintained on standard food pellets and water ad libitum. All animal care and procedures were in accordance with Institutional and National Institute of Health guidelines.

Diabetes was induced in rats by single intrafemoral injection of streptozotocin (Sigma Chemical Co., St. Louis, MO) freshly dissolved in 0.1 M citrate buffer, pH 4.5, under anesthesia. Streptozotocin was given at a dose of 55 mg/kg body weight 34 35 . The diabetic + IIH group received daily 2 doses (10 Unit/kg body weight) and control + IIH group received daily 2 doses (1.5 Unit/kg body weight) of regular human insulin (Actrapid) 36 . Diabetic + IIH and control + IIH group had daily two episodes of insulin-induced hypoglycemia for 10 days. Control rats were injected with citrate buffer. Glucose was measured by GOD-POD glucose estimation kit (Biolab Diagnostics Pvt Ltd). Rats were sacrificed by decapitation on the 10^th day of the experiment. The cerebellum was dissected out quickly over ice according to the procedure of Glowinski and Iversen 37 and the tissues collected were stored at -80°C until assayed.

Hypoglycemia impose alterations upon both the central (CNS) and peripheral (PNS) nervous systems which leads to brain damage and long-term cognitive impairment. The brain and other tissues require glucose in order to function properly. Neurotransmitters show significant alterations during hyperglycemia and cause degenerative changes in neurons of the central nervous system 41 42 . Severe hypoglycemia with brain dysfunction limits intensified therapy in patients with insulin dependent diabetes mellitus, despite evidence that such therapy reduces the risk of chronic complications of the disease 43 .

In our earlier studies, we reported the glutamate mediated excitotoxicity in the cerebellum of insulin induced hypoglycemic and streptozotocin induced diabetic rats 32 . In the present study, we have demonstrated the role of cholinergic receptors during recurrent hypoglycemia in diabetic rats. Experimental evidence indicate the involvement of the cerebellum in variety of human mental activities including language, attention, cognitive affective syndromes 44 and motor relearning 45 . The cerebellar vermis integrates and processes the inputs from the vestibular, visual and proprioceptive systems to coordinate muscle timing as a result of which the centre of gravity stays within the limits of stable upright standing 46 . Cerebellum participates in learning and coordination of anticipatory operations which are necessary for the effective and timely directing of cognitive and non-cognitive resources 47 . Diabetes mellitus has been reported to be accompanied by a number of behavioral and hormonal abnormalities, including reduced locomotor activity 48 . Acute hypo- and hyperglycemia have disruptive effects on the central nervous system 49 50 . Complications associated with diabetes involve neuronal damage which leads to altered neurotransmitter functions and reduced motor activity.

Glucose sensitive neurons organize and respond to changes in a number of hormonal, metabolic, transmitter, and peptide signals which involve the regulation of energy homeostasis and other biological functions 51 . Glucose deprivation causes neuronal death affecting the cognitive and memory ability. Hypoglycemia and glucose deprivation causes mitochondrial damage 52 . GLUT3 is one of the predominant glucose transporters located on neurons 53 . GLUT3 had its highest expression in brain and neural tissue hence being called the brain glucose transporter 54 . Our results shows an increased gene expression of GLUT3 expression in cerebellum of diabetic group and the hypoglycemic group showed a significant increase compared to diabetic group which shows that cerebellar glucose transport impairment is maximal during insulin induced hypoglycemia leading to neuronal dysfunction. Recent study demonstrated decreased glucose uptake-to-utilization ratio and increased leak of glucose in the cerebellum which showed that the cerebellum is not invariably resistant to hypoglycemia 55 . Disorders in the transport and metabolism of glucose are an important signal for triggering the apoptotic cascade 56 .

Changes in acetylcholine receptor have been implicated in the pathophysiology of many major diseases of the central nervous system. As in brain injury associated with ischaemia and neurodegenerative conditions, altered neurotransmitter action appears to play a role in hypoglycemic brain injury 7 8 9 . Cholinergic receptors activate a multitude of signaling pathways important for modulating neuronal excitability, synaptic plasticity and feedback regulation of ACh release 16 . The Muscarinic acetylcholine receptors are widely distributed throughout the body, but are predominantly expressed within the parasympathetic nervous system and exert both excitatory and inhibitory control over central and peripheral tissues. In the present study, enhanced muscarinic M1 and M3 receptor binding in the cerebellum of insulin induced hypoglycemia in both diabetic and nondiabetic rats along with increased AchE activity and decreased ChAT expression shows altered acetylcholine metabolism in the cerebellum. Cognitive deficits are reported to be connected with impairments of the cholinergic system 57 . Muscarinic acetylcholine receptor subtypes together with the activity of the cholinesterases (ChEs), mediate facilitation or depression of synaptic transmission 58 and AChE activity has been found to determine the range of ACh concentrations. Previous reports shows that insulin-induced hypoglycemia in normothermic rats caused progressive neurological depression and differentially altered regional cerebral acetylcholine metabolism 59 .

Neuronal nicotinic cholinergic receptors are crucial to acetylcholine neurotransmission in CNS. Our results show a significant upregulation in α7nAchR gene expression induced by hypoglycemia in diabetes and control rats when compared to diabetic rats which is suggested to cause nicotinic receptor mediated dysfunction. α7nAChRs are located in brain areas important for cognition and dysfunction of α7nAChRs in cerebellum is associated with cholinergic deficit. In the cerebellum, nicotinic acetylcholine receptors mediate the release of glutamate 17 , GABA 18 and norepinephrine 20 . Thus, these receptors significantly influence the activity within the cerebellar circuitry, and any deregulation of this activity contributes to functional deficit.

Acetylcholine mediated neurotransmission is involved in neuromuscular functions cerebellar dysfunction is associated with poor fine motor skills and hypotonia 13 . Acetylcholinesterase is critical for ensuring normal synaptic transmission. It is found that patients who recover from severe hypoglycemia are left with difficulties in cognition, particularly short-term memory, out of proportion to gross motor disability 4 . Our results showed an increased acetylcholine esterase activity in the muscle of hypoglycemic rats compared to diabetic group which shows neuromuscular dysfunction mediated by acetylcholine in the muscle of experimental rats. Up regulation of glutamate receptor activity causing motor dysfunction associated with cerebellum was demonstrated by the rotarod test in our previous studies 60 . Integrity of the neuromuscular junction is altered during hypoglycemia as reported by Thomareis et al 61 . It is observed that there is occurrence of seizures in hypoglycemic state which is due to the decreased glucose for the brain cells to function 62 .

To summarise, our results shows dysfunction of cerebellar cholinergic receptor due to impaired neuronal glucose transport in the cerebellum during recurrent hypoglycemia in diabetic rats. The receptor analysis and gene expression studies along with muscle acetylcholine esterase activity implicate a role for acetylcholine and cholinergic receptors in the modulation of neuronal network excitability and neuromuscular dysfunction associated with hypoglycemia. Our results supports previous reports that cerebellum is not spared during recurrent hypoglycemia in diabetes. These neurofunctional deficits are one of the key contributors to motor deficits and cellular stress associated with hypoglycemia in diabetes which is suggested to cause more damage at molecular level than hyperglycemia.

AchE: acetycholine esterase; ChAT: choline acetyltransferase; α7nAchR: alpha7 nicotinic acetylcholine receptor; QNB: Quinuclidinyl benzilate; L: benzilic - 4,4'; DAMP: 4- deoxy acetyl methyl piperidine mustard; D + IIH: Insulin induced hypoglycemia in diabetes; C + IIH: Insulin induced hypoglycemia in Control.

The authors declare that they have no competing interests.

SA and CSP designed research. SA and PKT carried out experiments and drafted the manuscript. JM and ATR helped in experiments. All authors read and approved the final manuscript.